Antimicrobial sulfonamide derivatives of lipopeptide antibiotics

ABSTRACT

The present invention provides antimicrobial sulfonamide derivatives of lipopeptide antibiotics, pharmaceutical compositions of antimicrobial sulfonamide derivatives, methods for making antimicrobial sulfonamide derivatives, methods for inhibiting microbial growth with antimicrobial sulfonamide derivatives and methods for treating or preventing microbial infections in a subject with antimicrobial sulfonamide derivatives. Antimicrobial sulfonamide derivatives are generally an amino core antibiotic that has been further modified with a lipophilic sulfonyl group.

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/760,328, filed Jan. 12, 2001, now U.S. Pat. No. 6,511,962which claimed the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 60/219,059, filed Jul. 17, 2000 and U.S. ProvisionalApplication No. 60/220,950, filed Jul. 26, 2000. The above applicationsare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to novel antibiotics and antimicrobialagents. More particularly, the present invention relates toantimicrobial sulfonamide derivatives of lipopeptide antibiotics.

BACKGROUND OF THE INVENTION

An important class of antibiotics that inhibit Gram-positive bacteriaare the acidic lipopeptide antibiotics. Generally, acidic lipopeptideantibiotics consist of either a cyclic peptide core or a cyclicdepsipeptide core acylated with a lipophilic fragment. The lipophilicfragment, typically an unsaturated fatty acid, may be of varying length.Frequently, the antibiotic activity of lipopeptide antibiotics isrelated to the length of the lipophilic fragment.

Examples of acidic lipopeptide antibiotics include, but are not limitedto, laspartomycin (Umezawa et al., U.S. Pat. No. 3,639,582; Naganawa etal., 1968, J. Antibiot., 21, 55; Naganawa et al., 1970, J. Antibiot.,23, 423), zaomycin (Kuroya, 1960, Antibiotics Ann., 194; Kuroya,Japanese Patent No. 8150), crystallomycin (Gauze et al., 1957,Antibiotiki, 2, 9), aspartocin (Shay et al., 1960, Antibiotics Annual,194; Hausman et al., 1964, Antimicrob. Ag. Chemother., 352; Hausman etal., 1969, J. Antibiot., 22, 207; Martin et al., 1960, J. Am. Chem.Soc., 2079), amphomycin (Bodanszky et. al., 1973, J. Am. Chem. Soc., 95,2352), glumamycin (Fujino et al., 1965, Bull. Chem. Soc. Jap., 38, 515),brevistin (Shoji et al., 1976, J. Antibiotics, 29, 380), cerexin A(Shoji et al., 1976, J. Antibiotics, 29, 1268), cerexin B (Shoji et al.,1976, J. Antibiotics, 29, 1275), Antibiotic A-30912 (Hoehn et al., U.S.Pat. No. 5,039,789), Antibiotic A-1437 (Hammann et al., EP 0 629 636 B1;Lattrell et al., U.S. Pat. No. 5,629,288), Antibiotic A-54145 (Fukada etal., U.S. Pat. No. 5,039,789; Boeck et al., 1990, J. Antibiotics, 43,587), Antibiotic A-21978C (Debono et al., 1988, J. Antibiotics, 41,1093) and tsushimycin (Shoji et. al., 1968, J. Antibiot., 21, 439). Seealso Berdy, “CRC Handbook of Antibiotic Compounds,” Volume IV, Part 1,pages 313-327, CRC Press, Boca Raton, Fla., (1980); Korzybinski et al.,“Antibiotics-Origin Nature and Properties,” Vol. 1, Pergamon Press, pp.397-401 and 404-408, New York, N.Y. (1967).

Despite the efficacy of lipopeptide antibiotics against Gram-positivebacteria, the medicinal chemistry of these antibiotics has remainedlargely unexplored. However, given the recent dramatic rise ofantibiotic-resistant pathogens and infectious diseases, caused in partby frequent over use of antibiotics, the need for new antimicrobialagents is urgent (Cohen et al., 1992, Science, 257, 1050-1055).Methicillin resistant bacteria are a particular problem, since they arealso frequently resistant to a wide variety of other antibiotics(Yoshida et al., U.S. Pat. No. 5,171,836). Gram-positive bacteria, suchas Staphylococci, which cause persistent infections, are especiallydangerous when methicillin resistant. Even more alarmingly,vancomycin-resistant strains of Enterococcus faecium have been observed(Moellering, 1990, Clin. Microbiol. Rev., 3, 46). Strains resistant tovancomycin pose a serious health threat to society since vancomycin isthe antibiotic of last resort for several harmful pathogens.

Thus, there is a need to explore the medicinal chemistry of lipopeptideantibiotics to develop novel antimicrobial agents. The discovery of newlipopeptide antibiotics will increase the repertoire of antibioticsavailable to combat pathogens resistant to currently availableantibiotics.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides antimicrobial sulfonamidederivatives of lipopeptide antibiotics. The sulfonamide derivativesgenerally comprise the peptidic portion of a lipopeptide antibiotic(“core antibiotic” or “core cyclic peptide”) and a lipophilic moiety.The lipophilic moiety is linked to the amino core antibiotic or aminocore cyclic peptide, either directly or by way of an optionalintervening linker. The lipophilic moiety and core antibiotic or corecyclic peptide are connected by a linkage containing at least onesulfonamide group. When an optional linker is used, the sulfonamidelinkage may be between (i) the linker and the core antibiotic or corecyclic peptide; (ii) the lipophilic moiety and the linker; or both (i)and (ii) above.

The core antibiotic is the molecule obtained by enzymatic removal of thelipophilic moiety of a lipopeptide antibiotic, typically with adeacylase such as that produced by Actinoplanes utahensis (NRRL 12052).Lipopeptide antibiotics which may be used to provide a core antibiotic,include by way of example and not limitation, laspartomycin, zaomycin,crystallomycin, aspartocin, amphomycin, glumamycin, brevistin, cerexinA, cerexin B, Antibiotic A-30912, Antibiotic A-1437, Antibiotic A-54145,Antibiotic A-21978C and tsushimycin. Those of skill in the art willrecognize that for some of these lipopeptide antibiotics, removal of thelipophilic portion via enzymatic deacylation yields a cyclic peptide ordepsipeptide having one or more additional amino acids attached thereto.In some instances these additional exocyclic amino acids may benecessary for activity and should not be removed by further enzymaticdegradation. When enzymatic deacylation yields a cyclic peptide ordepsipeptide having additional exocyclic amino acids attached thereto,the “core antibiotic” includes the exocyclic amino acids. The corecyclic peptide is a cyclic peptide or depsipeptide with no exocyclicamino acids. In some situations the core cyclic peptide and the coreantibiotic may refer to the same molecule (e.g., Antibiotic A-30912).

The lipophilic moiety may be a saturated or unsaturated fatty acid. Thefatty acid may be branched or a straight-chain. Unsaturated fatty acidsmay be mono, di, tri, or polyunsaturated. The lipophilic moiety may alsobe substituted with heteroatoms, aryl groups, heteroaryl groups and thelike and may also be mono, di, tri, or polyunsaturated. In somesituations the lipophilic moiety may consist of a aryl group, arylarylgroup, biaryl group, heteroaryl group and the like.

The optional linker may comprise virtually any molecule capable oflinking the lipophilic moiety to the core antibiotic. Linkers suitablefor use are typically at least bi-functional, having one functionalgroup capable of forming a covalent linkage with an exocyclic amine ofthe core antibiotic and another functional group capable of forming acovalent linkage with a complementary functional group on a precursor ofthe lipophilic moiety. At least one of the linkages formed andoptionally both of the linkages formed are a sulfonamide linkage.

A wide variety of linkers suitable for spacing the lipophilic group fromthe core antibiotic or core cyclic peptide of a lipopeptide antibioticare known in the art and include by way of example and not limitation,linkers that contain alkyl, heteroalkyl, acyclic heteroatomic bridges,aryl, arylaryl, arylalkyl, heteroaryl, heteroaryl-heteroaryl,substituted heteroaryl-heteroaryl, heteroarylalkyl,heteroaryl-heteroalkyl and the like. Linkers may include single, double,triple or aromatic carbon-carbon bonds, nitrogen-nitrogen bonds,carbon-nitrogen, carbon-oxygen bonds and/or carbon-sulfur bonds andinclude functionalities such as carbonyls, ethers, thioethers,carboxamides, sulfonamides, ureas, urethanes, hydrazines, etc.

The linker may be flexible or rigid. Rigid linkers include, for example,polyunsaturated alkyl, aryl, biaryl, heteroaryl, etc. Flexible linkersinclude, for example, a flexible peptide such as Gly-Gly-Gly or aflexible saturated alkanyl or heteroalkanyl. The linker may behydrophilic or hydrophobic. Hydrophilic linkers include, for example,polyalcohols or polyethers such as polyalkyleneglycols. Hydrophobiclinkers may be, for example, alkyls or aryls.

In another aspect, the present invention provides methods for makingantimicrobial sulfonamide derivatives. Generally the methods involveassembling three fragments of the sulfonamide derivatives: the aminocore antibiotic or amino core cyclic peptide, the optional linker andthe lipophilic moiety in any convenient order. The only requirement isthat the precursor of the lipophilic moiety and/or linker bearappropriate functional groups such that the assembly of the fragmentsresult in the formation of at least one sulfonamide linkage. Forexample, a lipophilic sulfonyl derivative may be covalently attached toa amino core antibiotic or amino core cyclic peptide. As anotherexample, a linker may be first covalently attached to an amino coreantibiotic or amino core cyclic peptide and then a lipophilic sulfonylattached therefore. As still another example, a lipophilic sulfonylderivative may be covalently attached to a linker and the resultantlipophilic linker covalently attached to a amino core antibiotic oramino core cyclic peptide.

In still another aspect, the present invention provides pharmaceuticalcompositions comprising antimicrobial sulfonamide derivatives of theinvention. The pharmaceutical compositions generally comprise one ormore antimicrobial sulfonamide derivatives of the invention, (or saltsthereof) and an adjuvant, carrier, excipient or diluent. Thepharmaceutical composition may be formulated for environmental use, suchas for application on plants, for vetinary use or for pharmaceuticaluse. The choice of adjuvant, carrier, excipient or diluents will dependon the particular application.

In yet another aspect, the present invention provides methods ofinhibiting the growth of microbes such as Gram-positive bacteria. Themethod generally involves contacting a microbe with one or moreantimicrobial sulfonamide derivatives of the invention or a salt thereofor a pharmaceutical composition thereof in an amount effective toinhibit the growth of the microbe. The method may be practical toachieve a bacteriostatic effect, where the growth of the microbe isinhibited, or to achieve a bactericidal effect, where the microbe iskilled.

In a final aspect, the present invention provides methods for treatingand/or preventing microbial infections in a subject such as a human, aplant or an animal. The methods generally involve administering to asubject one or more of the antimicrobial sulfonamide derivatives, saltsor compositions of the invention in an amount effective to treat orprevent a microbial infection in the human, animal or plant. Theantimicrobial sulfonamide derivatives, salts or compositions may beadministered systemically or applied topically, depending on the natureof the microbial infection.

DETAILED DESCRIPTION OF THE INVENTION

4.1 Definitions

As used herein, the following terms shall have the following meanings:

“Core cyclic peptide” refers to the desamino portion cyclic peptide orcyclic depsipeptide portion of a lipopeptide antibiotic that remainsafter the lipophilic portion of a lipopeptide antibiotic, including anyexocyclic amino acids, has been removed. As illustrative examples, thecore cyclic peptides derived from the lipopeptide antibioticslaspartomycin (2), aspartocin (4), antibiotic A-21978C (6), antibioticA-54145 (8), antibiotic A-30912A (10), antibiotic A-30912B (12),antibiotic A-30912D (14) and antibiotic A-30912H (16) are depictedbelow:

As used herein, “core cyclic peptide” includes both conventional cyclicpeptides, such as the cyclic peptides derived from aspartocin (4) aswell as cyclic depsipeptides such as the cyclic depsipeptide derivedfrom Antibiotic A-21978C (6) and Antibiotic A-54145 (8). The dashedlines in structures 2, 4, 6, 8, 10, 12, 14 and 16 indicate points ofattachment of the amido lipophilic portion of the parent lipopeptideantibiotic or, for those lipopeptide antibiotics in which the aminolipophilic portion is linked to the core peptide via interveningexocyclic amino acid residues, the dashed lines indicate the point ofattachment of the amino group of exocyclic amino acid residues.

“Amino core cyclic peptide” refers to a core cyclic peptide as definedabove where a primary amino group is attached to the dashed lines instructures 2, 4, 6, 8, 10, 12, 14 and 16.

“Core antibiotic” refers to the des-amino peptide portion of thelipopeptide antibiotic that remains after cleavage of the lipophilicfragment. As illustrative examples, the core antibiotics derived fromthe lipopeptide antibiotics laspartomycin 18, aspartocin 20, antibioticA-21978C 22, antibiotic A-54145 24 and Antibiotic A-30912A 26 aredepicted below:

In structures 18, 20, 22, 24 and 26, the dashed lines indicate the pointof attachment of the amine group that joins the lipophilic moiety andthe core antibiotic in the naturally occurring antibiotic. Thestructures of core antibiotics derived from Antibiotic A-30912B,Antibiotic A-30912D and Antibiotic A-30912H will be apparent to those ofskill in the art.

“Amino core antibiotic” refers to a core antibiotic as defined abovewhere a primary amino group is attached to the dashed lines instructures 18, 20, 22, 24 and 26.

“FMOC derivative of the core antibiotic of aspartocin” refers to thefollowing compound:

“t-butyl derivative of the core antibiotic of laspartomycin” refers tothe following compound:

As will be recognized by those of skill in the art, in somecircumstances, the core antibiotic and core cyclic peptide of alipopeptide antibiotic may be the same (see, e.g., the core cyclicpeptide 10 and core antibiotic 26 derived from antibiotic A-30912A).Also, the core antibiotic and the core cyclic peptide may be different(see, e.g., the core cyclic peptide 2 and core antibiotic 18 derivedfrom laspartomycin). Also, in some instances, the amino core antibiotic,which may include an exocyclic amino acid or peptide, may be furtherdeacylated further to yield the corresponding amino core cyclic peptide.For example, deacylation of laspartomycin with a deacylase produced byfermentation of Actinoplanes utahensis (NRRL 12052) yields both theamino core cyclic peptide of laspartomycin and the amino core antibioticof laspartomycin.

“A-30912” refers to all naturally occurring A-30912 compounds. Whenreference to a specific A-30912 compound or nucleus is intended, then aspecific designation such as A-30912 A, A-30912 B, A-30912 C, etc. isused.

“A-21978C” refers to all naturally occurring A-21978C compounds and isintended to include anhydro and isomeric forms (Baker et al., U.S. Pat.No. 5,912,225). When reference to a specific A-21978C compound ornucleus is intended, then a specific designation such as anhydro- andisomer, etc. is used.

“A-54145” refers to all naturally occurring A-54145 compounds. Whenreference to a specific A-54145 compound or nucleus is intended, then aspecific designation is used.

“Alkyl” refers to a saturated or unsaturated, branched, straight-chainor cyclic monovalent hydrocarbon group derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane, alkene oralkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl (allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

The term “alkyl” is specifically intended to include groups having anydegree or level of saturation, i.e., groups having exclusively singlecarbon-carbon bonds, groups having one or more double carbon-carbonbonds, groups having one or more triple carbon-carbon bonds and groupshaving mixtures of single, double and triple carbon-carbon bonds. Wherea specific level of saturation is intended, the expressions “alkanyl,”“alkenyl,” and “alkynyl” are used.

“Alkanyl” refers to a saturated branched, straight-chain or cyclic alkylgroup. Typical alkanyl groups include, but are not limited to, methanyl;ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl),cyclopropan-1-yl, etc.; butyanyls such as butan-1-yl, butan-2-yl(sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl(t-butyl), cyclobutan-1-yl, etc.; and the like.

“Alkenyl” refers to an unsaturated branched, straight-chain or cyclicalkyl group having at least one carbon-carbon double bond derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkene. The group may be in either the cis or trans conformation aboutthe double bond(s). Typical alkenyl groups include, but are not limitedto, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl;cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl,2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl,cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like.

“Alkynyl” refers to an unsaturated branched, straight-chain or cyclicalkyl group having at least one carbon-carbon triple bond derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkyne. Typical alkynyl groups include, but are not limited to, ethynyl;propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such asbut-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

“Aryl” refers to a monovalent aromatic hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like.

“Arylaryl” refers to a monovalent hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a ring systemin which two or more identical or non-identical parent aromatic ringsystems are joined directly together by a single bond, where the numberof such direct ring junctions is one less than the number of parentaromatic ring systems involved. Typical arylaryl groups include, but arenot limited to, biphenyl, triphenyl, phenyl-naphthyl, binaphthyl,biphenyl-naphthyl, and the like. Where the number of carbon atoms in anarylaryl group are specified, the numbers refer to the carbon atomscomprising each parent aromatic ring. For example, (C₅-C₁₄) arylaryl isan arylaryl group in which each aromatic ring comprises from 5 to 14carbons, e.g., biphenyl, triphenyl, binaphthyl, phenylnaphthyl, etc.Preferably, each parent aromatic ring system of an arylaryl group isindependently a (C₅-C₁₄) aromatic, more preferably a (C₅-C₁₀) aromatic.Also preferred are arylaryl groups in which all of the parent aromaticring systems are identical, e.g., biphenyl, triphenyl, binaphthyl,trinaphthyl, etc.

“Biaryl” refers to an arylaryl group having two identical parentaromatic systems joined directly together by a single bond. Typicalbiaryl groups include, but are not limited to, biphenyl, binaphthyl,bianthracyl, and the like. Preferably, the aromatic ring systems are(C₅-C₁₄) aromatic rings, more preferably (C₅-C₁₀) aromatic rings. Aparticularly preferred biaryl group is biphenyl.

“Arylalkyl” refers to an acyclic alkyl group in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl group. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. Where specific alkyl moieties are intended, the nomenclaturearylalkanyl, arylakenyl and/or arylalkynyl is used. In preferredembodiments, the arylalkyl group is (C₆-C₂₀) arylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₆) andthe aryl moiety is (C₅-C₁₄). In particularly preferred embodiments thearylalkyl group is (C₆-C₁₃), e.g., the alkanyl, alkenyl or alkynylmoiety of the arylalkyl group is (C₁-C₃) and the aryl moiety is(C₅-C₁₀).

“Heteroaryl” refers to a monovalent heteroaromatic group derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like.

“Heteroarylalkyl” refers to an acyclic alkyl group in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or Sp³carbon atom, is replaced with a heteroaryl group. Where specific alkylmoieties are intended, the nomenclature heteroarylalkanyl,heteroarylakenyl and/or heterorylalkynyl is used.

“Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, indane, indene, phenalene, etc. Typical parent aromaticring systems include, but are not limited to, aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexalene, as-indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene,and the like.

“Parent Heteroaromatic Ring System” refers to a parent aromatic ringsystem in which one or more carbon atoms (and any associated hydrogenatoms) are each independently replaced with the same or differentheteroatom. Typical heteratoms to replace the carbon atoms include, butare not limited to, N, P, O, S, Si, etc. (Including and associatedhydrogen or other atoms). Specifically included within the definition of“parent heteroaromatic ring systems” are fused ring systems in which oneor more of the rings are aromatic and one or more of the rings aresaturated or unsaturated, such as, for example, arsindole, chromane,chromene, indole, indoline, xanthene, etc. Typical parent heteroaromaticring systems include, but are not limited to, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like.

“Pharmaceutically acceptable salt” refers to a salt of a compound of theinvention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. Such saltsinclude: (1) acid addition salts, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike.

Reference will now be made in detail to preferred embodiments of theinvention. While the invention will be described in conjunction withpreferred embodiments, it should be understood that it is not intendedto limit the invention to these preferred embodiments. To the contrary,it is intended to cover alternatives, modifications, and equivalents asmay be included within the spirit and scope of the invention as definedby the appended claims.

4.2 The Invention

The present invention provides antimicrobial sulfonamide derivatives,pharmaceutical compositions comprising antimicrobial sulfonamidederivatives, methods for making antimicrobial sulfonamide derivatives,methods for inhibiting microbial growth with antimicrobial sulfonamidederivatives and methods for treating or preventing microbial infectionsin a subject with antimicrobial sulfonamide derivatives.

Examples of acidic lipopeptide antibiotics that may be converted toantimicrobial sulfonamide derivatives include, but are not limited to,laspartomycin (Umezawa et al., U.S. Pat. No. 3,639,582; Naganawa et al.,1968, J. Antibiot., 21, 55; Naganawa et al., 1970, J. Antibiot., 23,423), zaomycin (Kuroya, 1960, Antibiotics Ann., 194; Kuroya, JapanesePatent No. 8150), crystallomycin (Gauze et al., 1957, Antibiotiki, 2,9), aspartocin (Shay et al., 1960, Antibiotics Annual, 194; Hausman etal., 1964, Antimicrob. Ag. Chemother., 352; Hausman et al., 1969, J.Antibiot., 22, 207; Martin et al., 1960, J. Am. Chem. Soc., 2079),amphomycin (Bodanszky et. al., 1973, J. Am. Chem. Soc., 95, 2352),glumamycin (Fujino et al., 1965, Bull. Chem. Soc. Jap., 38, 515),brevistin (Shoji et al., 1976, J. Antibiotics, 29, 380), cerexin A(Shoji et al., 1976, J. Antibiotics, 29, 1268), cerexin B (Shoji et al.,1976, J. Antibiotics, 29, 1275), daptomycin (Debono et. al., 1988, J.Antibiotics, 41, 1093), Antibiotic A-30912 (Hoehn et al., U.S. Pat. No.5,039,789), Antibiotic A-1437 (Hammann et al., EP 0 629 636 B1; Lattrellet al., U.S. Pat. No. 5,629,288), Antibiotic A-54145 (Fukada et al.,U.S. Pat. No. 5,039,789; Boeck et al., 1990, J. Antibiotics, 43, 587),Antibiotic A-21978C (Debono et al., 1988, J. Antibiotics, 41, 1093) andtsushimycin (Shoji et. al., 1968, J. Antibiot., 21, 439).

4.2.1 Antimicrobial Sulfonamide Derivatives

Antimicrobial sulfonamide derivatives of the present invention offersome significant advantages over traditional antibiotics. Antimicrobialsulfonamide derivatives are generally active against many gram positivebacteria. More importantly, antimicrobial sulfonamide derivatives of thepresent invention may be effective against methicillin resistantbacteria and/or strains resistant to vancomycin. Thus, antimicrobialsulfonamide derivatives may inhibit or prevent growth of a number ofmicrobes generally resistant to known antibiotics. Further,antimicrobial sulfonamide derivatives may offer greater resistance tomicrobial proteases than the corresponding antimicrobial amidederivatives. Accordingly, use of antimicrobial sulfonamide derivativesmay be less likely to lead to the formation of antibiotic-resistantpathogens than conventional antimicrobial agents.

Those of skill in the art will appreciate that many of the compounds andcompound species described herein may exhibit the phenomena oftautomerism, conformational isomerism, geometric isomerism and/orstereoisomerism. As the formula drawings within the specification canrepresent only one of the possible tautomeric, conformational isomeric,enantiomeric or geometric isomeric forms, it should be understood thatthe invention encompasses any tautomeric, conformational isomeric,enantiomeric and/or geometric isomeric forms of the compounds having oneor more of the utilities described herein, as well as mixtures of thesevarious different forms.

The present invention provides antimicrobial sulfonamide derivativesaccording to structural formula (I):

Y—X—N(R⁴)(-L—X—N(R¹))_(m)—R  (I)

wherein:

Y is a lipophilic moiety;

each X is independently selected from the group consisting of —CO——SO₂—, —CS—, —PO—, —OP(O)—, —OC(O)—, —NHCO— and —N(R¹)CO— with theproviso that at least one X is —SO₂—;

m is 0 or 1;

L is a linker;

N is nitrogen;

R¹ and R⁴ are each independently selected from the group consisting ofhydrogen, (C₁-C₂₅) alkyl optionally substituted with one or more of thesame or different R² groups, (C₁-C₂₅) heteroalkyl optionally substitutedwith one or more of the same or different R² groups, (C₅-C₃₀) aryloptionally substituted with one or more of the same or different R²groups, (C₅-C₃₀) arylaryl optionally substituted with one or more of thesame or different R² groups, (C₅-C₃₀) biaryl optionally substituted withone or more of the same or different R² groups, five to thirty memberedheteroaryl optionally substituted with one or more of the same ordifferent R² groups, (C₆-C₃₀) arylalkyl optionally substituted with oneor more of the same or different R² groups and six to thirty memberedheteroarylalkyl optionally substituted with one or more of the same ordifferent R² groups;

each R² is independently selected from the group consisting of —OR³,—SR³, —NR³R³, —CN, —NO₂, —N₃, —C(O)OR³, —C(O)NR³R³, —C(S)NR³R³,—C(NR³)NR³R³, —CHO, —R³CO, —SO₂R³, —SOR³, —PO(OR³)₂, —PO(OR³), —CO₂H,—SO₃H, —PO₃H, halogen and trihalomethyl;

each R³ is independently selected from the group consisting of hydrogen,(C₁-C₆) alkyl, (C₅-C₁₀) aryl, five to sixteen membered heteroaryl,(C₆-C₁₆) arylalkyl and six to sixteen membered heteroarylalkyl; and

R is a core cyclic peptide or a core antibiotic of a lipopeptideantibiotic.

When m is 1 the antimicrobial sulfonamide derivatives of the inventionare represented by structural formula (II):

Y—X—N(R⁴)-L—X—N(R¹)—R  (II)

Here, the nitrogen atom covalently bonded to R¹ is directly attached tothe core cyclic peptide or core antibiotic of a lipopeptide antibiotic(see section 4.1). The nitrogen atom covalently bonded to R⁴ iscovalently bonded to both the sulfonyl group and the linker L.

In a preferred embodiment, R¹ and R⁴ are independently selected from thegroup consisting of hydrogen, (C₁-C₁₀) alkyl optionally substituted withone or more of the same or different R² groups, (C₁-C₁₀) heteroalkyloptionally substituted with one or more of the same or different R²groups, (C₅-C₁₅) aryl optionally substituted with one or more of thesame or different R² groups, (C₅-C₁₅) biaryl optionally substituted withone or more of the same or different R² groups, five to sixteen memberedheteroaryl optionally substituted with one or more of the same ordifferent R² groups, (C₆-C₁₆) arylalkyl optionally substituted with oneor more of the same or different R² groups and six to sixteen memberedheteroarylalkyl optionally substituted with one or more of the same ordifferent R² groups where R² is a substituent as defined above inFormula (I).

Preferably, R¹ and R⁴ are independently selected from the groupconsisting of hydrogen, (C₁-C₆) alkanyl optionally substituted with oneor more of the same or different R² groups, (C₃-C₇) alkenyl optionallysubstituted with one or more of the same or different R² groups, C₆ aryloptionally substituted with one or more of the same or different R²groups, C₁₂ biaryl optionally substituted with one or more of the sameor different R² groups, five to sixteen membered heteroaryl optionallysubstituted with one or more of the same or different R² groups,(C₆-C₁₀) arylalkyl optionally substituted with one or more of the sameor different R² groups and (C₆-C₁₀) heteroarylalkyl optionallysubstituted with one or more of the same or different R² groups, whereR² is a substituent as defined above in Formula (I). More preferably, R¹and R⁴ are independently selected from the group consisting of hydrogen,methyl, allyl, homoallyl, phenyl optionally substituted with one or moreof the same or different R² groups and benzyl optionally substitutedwith one or more of the same or different R² groups, where R² is asubstituent as defined above in Formula (I). Most preferably, R¹ and R⁴are hydrogen.

When the antimicrobial sulfonamide derivative is described by structuralformula (II) (i.e., Y—X—N(R⁴)-L—X—N(R¹)—R), the moiety X may be any kindof chemical functionality that can form a covalent bond with nitrogenwith the proviso that at least one X is —SO₂—. Preferably, X is —CO—,—SO₂—, —CS—, —PO—, —OPO—, —OC(O)—, —NHCO— or —NR⁵CO— where R⁷ isselected from the group consisting of hydrogen, (C₁-C₆) alkyl, (C₅-C₁₀)aryl, five to sixteen membered heteroaryl, (C₆-C₁₆) arylalkyl and six tosixteen membered heteroarylalkyl. More preferably, X is —CO— or —SO₂.

Connected to X—N(R¹) in the antimicrobial sulfonamide derivativedescribed by Formula (II) (i.e., Y—XN(R⁴)-L—X—N(R¹)—R) is a linker L anda nitrogen group (i.e., N(R⁴)). The nature of linker L may varyextensively. Thus, for example, L may be hydrophilic or hydrophobic,long or short, rigid, semirigid or flexible etc.

A wide variety of linkers L comprised of stable bonds suitable forspacing the lipophilic group Y from the core cyclic peptide or coreantibiotic of a lipopeptide antibiotic are known in the art, and includeby way of example and not limitation, linkers such as alkyl,heteroalkyl, acyclic heteroatomic bridges, aryl, arylaryl, arylalkyl,heteroaryl, heteroaryl-heteroaryl, substituted heteroaryl-heteroaryl,heteroarylalkyl, heteroaryl-heteroalkyl and the like. Thus, linker L mayinclude single, double, triple or aromatic carbon-carbon bonds,nitrogen-nitrogen bonds, carbon-nitrogen, carbon-oxygen bonds and/orcarbon-sulfur bonds, and may therefore include functionalities such ascarbonyls, ethers, thioethers, carboxamides, sulfonamides, ureas,urethanes, hydrazines, etc.

Choosing a suitable linker is within the capabilities of those of skillin the art. For example, where a rigid linker is desired, L may be arigid polyunsaturated alkyl or an aryl, biaryl, heteroaryl, etc. Where aflexible linker is desired, L may be a flexible peptide such asGly-Gly-Gly or a flexible saturated alkanyl or heteroalkanyl.Hydrophilic linkers may be, for example, polyalcohols or polyethers suchas polyalkyleneglycols. Hydrophobic linkers may be, for example, alkylsor aryls.

Some embodiments of N(R⁴)-L include, for example, compounds where L is—(CH₂)_(k)—, k is an integer between 1 and 8 and the correspondinganalogues where any suitable hydrogen is independently substituted withone or more of the same or different R² groups, where R² is defined asabove in Formula (I). Other embodiments of N(R⁴)-L include any aminoacid or peptide, which may be for example, a D or L α-amino acid, aβ-amino acid, a γ-amino acid, a dipeptide, a tripeptide or atetrapeptide comprised of any combination of amino acids preferably,α-amino acids). The polarity of the peptide bond in these peptides maybe either C→N or N→C.

In a preferred embodiment, when m is 1, L is selected from the groupconsisting of:

or a pharmaceutically acceptable salt or hydrate thereof, wherein:

n is 0, 1, 2 or 3;

each S¹ is independently selected from the group consisting of hydrogen,(C₁-C₁₀) alkyl optionally substituted with one or more of the same ordifferent R⁵ groups, (C₁-C₁₀) heteroalkyl optionally substituted withone or more of the same or different R⁵ groups, (C₅-C₁₀) aryl optionallysubstituted with one or more of the same or different R⁵ groups,(C₅-C₁₅) arylaryl optionally substituted with one or more of the same ordifferent R⁵ groups, (C₅-C₁₅) biaryl optionally substituted with one ormore of the same or different R⁵ groups, five to ten membered heteroaryloptionally substituted with one or more of the same or different R⁵groups, (C₆-C₁₆) arylalkyl optionally substituted with one or more ofthe same or different R⁵ groups and six to sixteen memberedheteroarylalkyl optionally substituted with one or more of the same ordifferent R⁵ groups;

each R⁵ is independently selected from the group consisting of —OR⁶,—SR⁶, —NR⁶R⁶, —CN, —NO₂, —N₃, —C(O)OR⁶, —C(O)NR⁶R⁶, —C(S)NR⁶R⁶,—C(NR⁶)NR⁶R⁶, —CHO, —R⁶CO, —SO₂R⁶, —SOR⁶, —PO(OR⁶)₂, —PO(OR⁶), —CO₂,—SO₃H, —PO₃H, halogen and trihalomethyl;

each R⁶ is independently selected from the group consisting of hydrogen,(C₁-C₆) alkyl, (C₅-C₁₀) aryl, five to ten membered heteroaryl, (C₆-C₁₆)arylalkyl and six to sixteen membered heteroarylalkyl; and

each K is independently selected from the group consisting of oxygen,nitrogen and sulfur.

In a preferred embodiment, each S¹ is independently a side chain of agenetically encoded a amino acid. Exemplary preferred embodiments, whereK is oxygen or nitrogen and S¹ is hydrogen, include the followingcompounds where R, R¹, R⁴ and Y are as previously defined:

In another embodiment, L is:

where n is as previously defined. Preferably, each S¹ is independentlythe side chain of a genetically encoded α amino acid.

In a preferred embodiment, n is 0 and S¹ is —CH₂—CO₂H, —CH₂—CH₂—CO₂H,—C(OH)H—CONH₂, —CH₂—CONH₂ or —CH₂—CH₂—CONH₂. In another preferredembodiment, n is 0 and S¹ is —CH₂—indol-2-yl or —CH₂-phenyl.

In one embodiment, n is 0, R⁴ is hydrogen and R is the core antibioticof aspartocin or the FMOC derivative of the core antibiotic ofaspartocin. In one preferred embodiment, S¹ is H and Y² is decan-yl. Inanother preferred embodiment, S¹ is —CH₂-phenyl and Y² is hexadecan-yl.

In another embodiment, n is 0, R⁴ is hydrogen and R is the coreantibiotic of laspartomycin. In one preferred embodiment, S¹ is—CH₂-indol-2-yl and Y² is hexadecan-yl. In another preferred embodiment,S¹ is —CH₂-phenyl and Y² is hexadecan-yl. In another preferredembodiment, S¹ is —CH₂-phenyl and Y² is decan-yl.

In another embodiment, n is 0, R⁴ is hydrogen and R is the core cyclicpeptide of laspartomycin. In one preferred embodiment, S¹ is—CH₂-indol-2-yl and Y² is hexadecan-yl.

In still another preferred embodiment, L is:

Preferably, S² and S² are the side chain of a genetically encoded αamino acid. In one embodiment, n is 1, S² is hydrogen, —CH₂-indol-2-yl,—CH₂—CONH₂ or —CH₂—CH₂—CONH₂ and S³ is —CH₂—CO₂H or —CH₂—CH₂—CO₂H. Inanother embodiment, n is 1, S² is —CH₂—CO₂H or —CH₂—CH₂—CO₂H and S³ is—C(OH)H—CONH₂.

In still another preferred embodiment L is:

Preferably, S², S³ and S⁴ are the side chain of a genetically encoded aamino acid. In one embodiment, n is 2, S² is —CH₂-indol-2-yl, S³ is—CH₂—CONH₂ or —CH₂—CH₂—CONH₂ and S⁴ is —CH₂—CO₂H or —CH₂—CH₂—CO₂H. Inanother embodiment, n is 2, S² is —CH₂-indol-2-yl, S³ is —CH₂—CO₂H orCH₂—CH₂—CO₂H— and S⁴ is —CH₂—CONH₂, —CH₂—CH₂—CONH₂ or —C(OH)H—CONH₂.

Preferably, R is the core cyclic peptide or core antibiotic oflaspartomycin, zaomycin, crystallomycin, aspartocin, amphomycin,glumamycin, brevistin, cerexin A, cerexin B, Antibiotic A-30912,Antibiotic A-1437, Antibiotic A-54145, Antibiotic A-21978C ortsushimycin. More preferably, R is the core antibiotic or core cyclicpeptide of laspartomycin, aspartocin, Antibiotic A-30912, AntibioticA-1437, Antibiotic A-54145 or Antibiotic A-21978C. Most preferably, R isthe core antibiotic or core cyclic peptide of laspartomycin oraspartocin.

In another preferred embodiment, m is 0. In this embodiment, theantimicrobial sulfonamide derivative of the invention is represented bystructural formula (III):

Y—SO₂N(R⁴)—R  (III)

Here, the nitrogen atom covalently bonded to R⁴ is directly attached tothe core cyclic peptide or core antibiotic of a lipopeptide antibiotic(see section 4.1). Preferred embodiments of R and R⁴ include thosedefined above. Particularly preferred embodiments include those where R⁴is hydrogen and/or R is the core antibiotic or core cyclic peptide oflaspartomycin or aspartocin. In one preferred embodiment of compounds ofFormula (III), Y is hexadec-yl, R⁴ is H and R is the core antibiotic oflaspartomycin or the t-butyl ester of the core antibiotic oflaspartomycin.

Generally, the lipophilic group Y will be hydrophobic and whensubstituted will be substituted with hydrophobic substituents. Those ofskill in the art will appreciate that the size and/or length of thelipophilic group will depend, in part, on the nature of fragments suchas L, X, R¹, R⁴ and R that comprise the antimicrobial sulfonamidederivative.

Preferably, the lipophilic group Y is selected from the group consistingof (C₂-C₃₀) alkyl optionally substituted with one or more of the same ordifferent R⁷ groups, (C₂-C₃₀) heteroalkyl optionally substituted withone or more of the same or different R⁷ groups, (C₅-C₃₀) aryl optionallysubstituted with one or more of the same or different R⁷ groups,(C₅-C₃₀) arylaryl optionally substituted with one or more of the same ordifferent R⁷ groups, (C₅-C₃₀) biaryl optionally substituted with one ormore of the same or different R⁷ groups, five to thirty memberedheteroaryl optionally substituted with one or more of the same ordifferent R⁷ groups, (C₆-C₃₀) arylalkyl optionally substituted with oneor more of the same or different R⁷ groups and six to thirty memberedheteroarylalkyl optionally substituted with one or more of the same ordifferent R⁷ groups;

each R⁷ is independently selected from the group consisting of —OR⁸,—SR⁸, —NR⁸R⁸, —CN, —NO₂, —N₃, —C(O)OR³, —C(O)NR⁸R⁸, C(S)NR⁸R⁸,—C(NR⁸)NR⁸R⁸, —CHO, —R⁸CO, —SO₂R⁸, —SOR⁸, —PO(OR⁸)₂, —PO(OR⁸), —CO₂H,—SO₃H, —PO₃H, halogen and trihalomethyl;

each R⁸ is independently selected from the group consisting of hydrogen,(C₁-C₆) alkyl, (C₅-C₁₀) aryl, five to fifteen membered heteroaryl,(C₆-C₁₆) arylalkyl and six to sixteen membered heteroarylalkyl.

The nature of the fragments such as L, X, R¹, R⁴ and R that comprise theantimicrobial sulfonamide derivative are particularly important indefining preferred embodiments of the lipophilic fragment Y. Those ofskill in the art will realize that preferred embodiments of Y willparticularly depend on the core cyclic peptide or core antibiotic and/orthe linker L. Accordingly, antimicrobial sulfonamide derivatives withdifferent core cyclic peptides or core antibiotics and/or linkers L willhave different preferred embodiments of the lipophilic fragment Y.

For example, when m is 0, X is —SO₂, R⁴ is previously defined, and R isthe core antibiotic of anhydro-Antibiotic-21987C orisomer-Antibiotic-21987 (or amino protected versions, thereof) preferredembodiments of Y include those described in Baker et al., U.S. Pat. No.5,912,226. Preferred embodiments of Y described in Baker et al., U.S.Pat. No. 5,912,226 are preferred embodiments in the current invention,when the lipophilic group Y is connected by a sulfonamide linkage to thecore antibiotic of anhydro-Antibiotic-21987C or isomer-Antibiotic-21987.

Alternatively, when m is 0, X is —SO₂—, R⁴ is as previously defined andR is the core antibiotic of Antibiotic-21987C (or an amino protectedversion thereof) preferred embodiments of the lipophilic fragment Yinclude those described by the Lilly group (Debono, U.S. Pat. No.RE32,333; Debono, U.S. Pat. No. RE32,311; Abbott et al., U.S. Pat. No.4,537,717; Abbott et al., U.S. Pat. No. 4,524,135; Abbott et al., U.S.Pat. No. 4,482,487; Debono U.S. Pat. No. 4,399,067; Debono U.S. Pat. No.4,396,543).

When m is 0, X is —SO₂—, R⁴ is as previously defined and R is the coreantibiotic of laspartomycin, preferred embodiments of Y include thosedescribed in Borders et al., U.S. patent application Ser. No.09/760,328.

When m is 0, X is —SO₂—, R⁴ is as previously defined and R is the coreantibiotic of Antibiotic A-1437 preferred embodiments of Y include thosedescribed in Lattrell et al., U.S. Pat. No. 5,629,288. Similarly, when mis 1, X is —SO₂— and —CO—, R¹ and R⁴ is as previously defined, L is thedes-amino derivative of asparagine and R is the core antibiotic ofAntibiotic A-1437, preferred embodiments of Y may be found in Lattrellet al., U.S. Pat. No. 5,629,288.

When m is 0, X is —SO₂—, R⁴ is as previously defined and R is the coreantibiotic of Antibiotic A54145, preferred embodiments of Y includethose described in Fukada et al., U.S. Pat. No. 5,028,590 and Fukada etal., U.S. Pat. No. 5,039,789.

It will be understood that the selection of Y is intimately related tothe structure of the core antibiotic or core cyclic peptide and linker.Given the structure of the core antibiotic or core cyclic peptide andlinker, selection of preferred embodiments of Y is well within thepurview of those of ordinary skill in the art given the examplesprovided above.

Particularly preferred antimicrobial sulfonamides include the followingcompounds.

R is the core antibiotic of laspartomycin

R is the core cyclic peptide of laspartomycin

R is the core antibiotic of laspartomycin

R is the core antibiotic of aspartocin

R is the FMOC derivative of the core antibiotic of aspartocin

R is the core antibiotic of aspartocin

4.2.2 Methods of Making Antimicrobial Sulfonamide Derivatives

Antimicrobial sulfonamide derivatives are preferably synthesized fromamino antibiotics or core cyclic peptides, which may be made by theapproaches described in Section 4.5 of this Application. Those of skillin the art will appreciate antimicrobial sulfonamide derivatives may besynthesized from a vast number of other different starting materials.

Starting materials useful for preparing antimicrobial sulfonamidederivatives from core antibiotics or core cyclic peptides are eithercommercially available or may be prepared by conventional syntheticmethods. A number of general synthetic approaches may be envisioned forconverting core antibiotics or core cyclic peptides to antimicrobialsulfonamide derivatives. These include, but are not limited to, theapproaches outlined in Schemes I-IV.

Y—SO₂X+HN(R⁴)-L—X—N(R¹)—R→Y—SO₂N(R⁴)-L—X—N(R¹)—R  Scheme I

In Scheme I, an activated lipophilic sulfonyl derivative (Y—SO₂X) isreacted with a free amino group (HN(R⁴)) attached to a linker (L) toform the sulfonamide linkage.

In Scheme II, an activated lipophilic sulfonyl derivative (Y—SO₂X) isreacted with a free amino group (HN(R¹)) attached to a core cyclicpeptide or to a core antibiotic to form the sulfonamide linkage.

Y—SO₂X+HN(R¹)—R→Y—SO₂—N(R¹)—R  Scheme II

In both Scheme I and Scheme II, activated lipophilic sulfonyl derivativeY—SO₂X takes the same form. Here, X may be an activated derivative, suchas, for example, halogen (e.g., fluoride, bromide, chloride or iodide)or active ester (e.g., pentaflourophenyl ester, N-hydroxy succinimideester, p-nitrophenylester, etc.). Preferably, X is ahydroxybenzotriazole ester or a sulfonyl chloride. Alternatively, X maybe OH, which is activated in situ by well known methods (aminium salts,uronium salts, carbodiimides, etc.) Methods for constructing thesulfonamide linkage are well-known to the skilled artisan and may befound in compendiums of synthetic methods (Beilstein Handbook of OrganicChemistry, Beilstein Institute of Organic Chemistry, Frankfurt, Germany;Feiser, L.; Feiser, M., Reagents for Organic Synthesis, Volumes 1-17,Wiley Interscience; Trost, B.; Fleming, I, Comprehensive OrganicSynthesis, Pergamon Press, 1991; Theilheimer's Synthetic Methods ofOrganic Chemistry, Volumes 1-45, Karger, 1991; Compendium of OrganicSynthetic Methods, Wiley Interscience, Volumes 1-7; March, J., AdvancedOrganic Chemistry, Wiley Interscience, 1991; Larock, R.; ComprehensiveOrganic Transformations, VCH Publishers, 1989; Paquette, L. (ed.),Encyclopedia of Reagents for Organic Synthesis, John Wiley & Sons,1995).

Those of skill in the art will appreciate that protection of reactivefunctionalities in Y, R¹, R⁴, L and R may be necessary for formation ofthe sulfonamide linkage. In the event that protection of Y, R¹, R⁴, Land R is necessary to form the sulfonamide bond, then deprotection of Y,R¹, R⁴, L and R will be necessary to provide the desired antimicrobialsulfonamide derivative. Methods for protection and deprotection oforganic groups are known to those of skill in the art and may be used asnecessary in the synthesis of antimicrobial sulfonamide derivatives (seee.g., Greene, T. W.; Wuts, P., Protective Groups in Organic Synthesis, ₃^(rd) edition, Wiley Interscience, 1999).

Y—SO₂N(R⁴)-L—X^(1′)+HN(R¹)—R→Y—SO₂N(R⁴)-L—X—N(R¹)—R  Scheme III

In Scheme III, lipophilic fragment Y and linker L, attached via asulfonamide bond, are covalently linked to, for example, a sulfonic orcarboxylic acid or an activated derivative thereof (i.e., X^(1′)) suchas an active ester or a halogen. Sulfonic acids and carboxylic acids maybe activated in situ by methods known to the skilled artisan (e.g.,uronium salts, phosphonium salts, carbodiimides, etc.). Methods formaking activated derivatives of carboxylic acids and sulfonic acids andreacting these derivatives with amines to form amides or sulfonamidesare known to those of skill in the art. Preferably, X^(1′) is ahydroxybenzotriazole ester or a chloro derivative of a sulfonic orcarboxylic acid. Linkages other than sulfonamides or carboxamides may beformed by methods known to those of skill in the art.

Y—SO₂N(R⁴)-L¹+L²—X—N(R¹)—R→Y—SO₂N(R⁴)-L—X—N(R¹)—R  Scheme IV

Scheme IV describes a convergent approach where Y—SO₂N(R⁴-L—X—N(R¹)—R issynthesized by combining two fragments (Y—SO₂N(R⁴)-L¹ and L²—X—N(R¹)—R)to form the antimicrobial sulfonamide derivative. Here L¹ and L² combineto form the linker L upon covalent bond formation. Such an approach maybe particularly useful when L is an oligomer such as a polyamide orpolyethers. Methods for combining oligomeric subunits such as ether oramide monomers, dimers, etc. are known to those of skill in the art(Bodanzsky, M., Principles of Peptide Synthesis; Springer Verlag, 1984;Bodanzsky, M., Practice of Peptide Synthesis; Springer Verlag, 1984).Fragment Y—SO₂N(R⁴)-L¹ may be made by forming a sulfonamide bond betweenY—SO₂X and HN(R⁴)-L¹ using methods described above. FragmentL²—X—N(R¹)—R may be made, as described above, by forming either asulfonamide bond or an amide between L²—X^(1′) and HN(R¹)—R, whereX^(1′) is either a carboxylic acid or a sulfonic acid or activatedderivatives thereof.

4.2.3 Methods of Obtaining Amino Core Cyclic Peptides and Amino CoreAntibiotics

Culturing microorganisms that yield acidic lipopeptide antibiotics maybe used to provide amino core antibiotics or amino core cyclic peptidesthat can be converted to antimicrobial sulfonamide derivatives.Microorganisms that synthesize acidic lipopeptide antibiotics are wellknown in the art (see e.g., Umezawa et al., U.S. Pat. No. 3,639,582;Debono et. al., 1988, J. Antibiotics, 41, 1093; Shay et al., 1960,Antibiotics Annual, 194; Hamill et al., U.S. Pat. No. 4,331,594; Hamillet al., U.S. Pat. No. 4,208,403; Hoehn et al., U.S. Pat. No. 4,024,245;Higgins et al., U.S. Pat. No. 4,024,246; Boeck et al., U.S. Pat. No.4,288,549; Boeck et al., U.S. Pat. No. 4,994,270; Boeck, U.S. Pat. No.4,977,083). Methods of growing inocula and inoculating culturing mediumare known to the skilled artisan and exemplary methods have beendescribed in the art. Id. Preferred media, times, temperatures and pHfor culturing microorganisms that yield acceptable amounts of acidiclipopeptide antibiotics are also known in the art. Id.

Preferably, acidic lipopeptide antibiotics produced by culturingmicroorganisms are purified from fermentation broth or culture mediumusing extractive methods. The acidic lipopeptide antibiotic may beisolated as either the free acid or the salt.

Alternatively, lipopeptide antibiotics may be purified and isolated fromfermentation broth or culture medium by any art-known technique such ashigh performance liquid chromatography, counter current extraction,centrifugation, filtration, precipitation, ion exchange chromatography,gel electrophoresis, affinity chromatography and the like, either beforeor after extractive purification using the methods of the currentinvention. The actual conditions used to purify a lipopeptideantibiotics will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, etc., and will be apparent to thosehaving skill in the

Generally, the lipophilic fragment of the acidic lipopeptide antibioticis enzymatically cleaved to provide the core antibiotic or core cyclicpeptide. Addition of an appropriate enzyme to the culture medium mayprovide the core antibiotic or core cyclic peptide directly, thusobviating the need to isolate the lipopeptide antibiotic (Kreuzman etal., U.S. Pat. No. 5,573,936). Alternatively, the lipopeptide antibioticmay be chemically deacylated to provide the core antibiotic or corecyclic peptide, although this method frequently leads to complexmixtures (see e.g., Shoji et al., J. Antibiotics 28, 764, 1975; Shoji etal., J. Antibiotics 29, 380, 1976; Shoji et al., J. Antibiotics 29,1268, 1976; Shoji et al., J. Antibiotics 29, 1275, 1976).

Preferably, isolated lipopeptide antibiotics are treated with an enzymethat removes at least the lipophilic fragment of the lipopeptideantibiotic. The enzyme may be, for example, a degradative enzyme such asa peptidase, esterase or thiolase, of which numerous examples exist inthe art (Chihahra et al., Agr. Biol. Chem. 38, 1767, 1974; Suzuki etal., J. Biochem., 56, 335, 1964; Konishi et al., U.S. Pat. No.5,079,148). Preferably, the enzyme is a deacylase (Abbot et al., U.S.Pat. No. 4,299,763; Abbot et al., U.S. Pat. No. 4,299,762; Abbot et al.,U.S. Pat. No. 4,293,490; Kleinschmidt et al., U.S. Pat. No. 3,150,059;Abbott et al., U.S. Pat. No. 4,293,482; Kimura et al., Japanese PatentNo. 4058/67; Kuwana et al., U.S. Pat. No. 4,050,989; Shoji et al., J.Antibiotics 28, 764, 1975; Shoji et al., J. Antibiotics 29, 380, 1976;Shoji et al., J. Antibiotics 29, 1268, 1976; Shoji et al., J.Antibiotics 29, 1275, 1976).

Preferably, the cleavage of lipopeptide antibiotics to core antibioticsor core cyclic peptides commences by culturing microorganisms thatproduces a deacylase. The lipopeptide antibiotic is then contacted withthe culture medium containing the deacylase. Microorganisms such asthose of the Actinoplanacae that produce deacylases are well known tothose of skill in the art. In a preferred embodiment, the microorganismActinoplanes utahensis (NRRL 12052) provides a deacylase that deacylatesmany lipopeptide antibiotics to yield core antibiotics or core cyclicpeptides (see e.g., Lattrell et al., U.S. Pat. No. 5,039,789; Fukuda etal., U.S. Pat. No. 5,039,789; Abbott et al., U.S. Pat. No. 4,320,054;Abbott et al., U.S. Pat. No. 4,537,717; Debono et al., U.S. Pat. No.4,293,483; Borders et al., U.S. Pat. No. 6,511,962).

Parent cultures of Actinoplanes utahensis (NRRL 12052) especiallysuitable for cleaving the lipophilic fragment of lipopeptide antibioticsmay be selected by methods known to those of skill in the art. Apreferred method for selecting a parent culture which provides improvedyields of core antibiotics or core cyclic peptides is described inSection 5.1

Growing inocula and inoculating culturing medium are also well known tothose of skill in the art and exemplary methods for Actinoplanesutahensis (NRRL 12052) are described in the art (see e.g., Boeck et al.,1988, J. Antibiot., 41, 1085; Debono et. al., 1988, J. Antibiotics, 41,1093; Lattrell et al., U.S. Pat. No. 5,039,789; Fukada et al., U.S. Pat.No. 5,039,789; Abbott et al., U.S. Pat. No. 4,320,054; Abbott et al.,U.S. Patent No. 4,537,717; Debono, U.S. Pat. No. 4,293,483) and Section5.1.

Any culturing medium which supports Actinoplanes utahensis (NRRL 12052)growth may be used and selection of such medium is within the capabilityof those of skill in the art. Representative examples of culturingmedium which supports Actinoplanes utahensis (NRRL 12052) growth may befound in the art (see e.g., Boeck et al., 1988, J. Antibiot., 41, 1085;Debono et. al., 1988, J. Antibiotics, 41, 1093; Lattrell et al., U.S.Pat. No. 5,039,789; Fukada et al., U.S. Pat. No. 5,039,789; Abbott etal., U.S. Pat. No. 4,320,054; Abbott et al., U.S. Pat. No. 4,537,717;Debono, U.S. Pat. No. 4,293,483) and Section 5.1.

Preferred media, times, temperatures and pH for culturing Actinoplanesutahensis (NRRL 12052) that provide good yields of the deacylase aredescribed in the art (see e.g., Boeck et al., 1988, J. Antibiot., 41,1085; Debono et. al., 1988, J. Antibiotics, 41, 1093; Lattrell et al.,U.S. Pat. No. 5,039,789; Fukada et al., U.S. Pat. No. 5,039,789; Abbottet al., U.S. Pat. No. 4,320,054; Abbott et al, U.S. Pat. No. 4,537,717;Debono, U.S. Pat. No. 4,293,483) and Section 5.2.3.

Representative procedures for deacylating lipopeptide antibiotics withActinoplanes utahensis (NRRL 12052) to provide core antibiotics or corecyclic peptides may be found in the art (see e.g., Boeck et al., 1988,J. Antibiot., 41, 1085; Debono et. al., 1988, J. Antibiotics, 41, 1093;Lattrell et al., U.S. Pat. No. 5,039,789; Fukada et al., U.S. Pat. No.5,039,789; Abbott et al., U.S. Pat. No. 4,320,054; Abbott et al., U.S.Pat. No. 4,537,717; Debono, U.S. Pat. No. 4,293,483) and Example 4.

Although, deacylation of lipopeptide antibiotics such as aspartocin,A-30912, A-21978C and laspartomycin with Actinoplanes utahensis (NRRL12052) has been successful it should be pointed out that otherprocedures may be necessary for other lipopeptide antibiotics. Enzymespossess a high degree of specificity and slight differences in thepeptide moiety or the lipophilic fragment may have a profound effect onthe rate of deacylation. Further, in some situations (i.e., AntibioticA-30912 and Antibiotic A-21978C) the lipophilic fragment may beselectively removed to provide the core antibiotic. In other situations(i.e., laspartomycin) cleavage of the lipophilic fragment is accompaniedby hydrolysis of exocyclic peptide bonds to provide the core cyclicpeptide. Thus, deacylation of lipopeptide antibiotics with a deacylasemay provide a number of different peptide products.

Alternatively, core antibiotics or core cyclic peptides may be producedby methods known in the art for synthesizing peptides. For example,linear peptides may be prepared using conventional solution phase orsolid phase peptide synthesis and then cyclized.

Core antibiotics or core cyclic peptides may be purified and isolatedfrom either fermentation broth or synthetic reaction mixtures by anyart-known technique such as high performance liquid chromatography,counter current extraction, centrifugation, filtration, precipitation,ion exchange chromatography, gel electrophoresis, affinitychromatography and the like. The actual conditions used to purify aparticular core antibiotic or core cyclic peptide will depend, in part,on factors such as net charge, hydrophobicity, hydrophilicity, etc. andwill be apparent to those having skill in the art. 4.2.4 Activity

Generally, active antimicrobial sulfonamide derivatives of the inventionare identified using in vitro screening assay. Indeed, in many instancesthe antimicrobial sulfonamide derivatives of the invention will be usedin vitro as preservatives, topical antimicrobial treatments, etc.Additionally, despite certain apparent limitations of in vitrosusceptibility tests, clinical data indicate that a good correlationexists between minimal inhibitory concentration (MIC) test results andin vivo efficacy of antibiotic compounds (Murray, 1994, AntimicrobialSusceptibility Testing, Poupard et al., eds., Plenum Press, NY; Knudsenet al., 1995, Antimicrob. Agents Chemother. 39(6):1253-1258). Thus,isolated antimicrobial sulfonamide derivatives useful for treatinginfections and diseases related thereto are also conveniently identifiedby demonstrated in vitro antimicrobial activity against specifiedmicrobial targets.

Generally, the in vitro antimicrobial activity of antimicrobial agentsis tested using standard NCCLS bacterial inhibition assays, or MIC tests(see, National Committee on Clinical Laboratory Standards “PerformanceStandards for Antimicrobial Susceptibility Testing,” NCCLS DocumentM100-S5 Vol. 14, No. 16, December 1994; “Methods for dilutionantimicrobial susceptibility test for bacteria that growaerobically—Third Edition,” Approved Standard M7-A3, National Committeefor Clinical Standards, Villanova, Pa.).

Alternatively, the antimicrobial sulfonamide derivatives of theinvention may be assessed for antimicrobial activity using in vivomodels. Again, such models are well-known in the art.

It will be appreciated that other assays, as are well known in the artor that will become apparent to those having skill in the art uponreview of this disclosure, may also be used to identify activeantimicrobial sulfonamide derivatives of the invention. Such assaysinclude, for example, the assay described in Lehrer et al., 1988, J.Immunol. Methods 108:153 and Steinberg et al., “Designer Assays forAntimicrobial Peptides: Disputing the ‘One Size Fits All’ Theory,” In:Antibacterial Peptide Protocols, Shafer, Ed., Humana Press, NJ.

Generally, antimicrobial sulfonamide derivatives of the invention willexhibit MICs of less than about 64 μg/mL, usually less than about 32μg/mL, preferably less than about 16 μg/mL and most preferably less thanabout 4 μg/mL. The antimicrobial sulfonamide derivatives of theinvention may also exhibit antifungal activity, having MICs of about 50μg/mL or less against a variety of fungi in standard in vitro assays.

Of course, compounds having MICs on the low end of these ranges, or evenlower, are preferred. Most preferred for use in treating or preventingsystemic infections are antimicrobial sulfonamide derivatives thatexhibit significant antimicrobial activity (i.e., less than 4 μg/mL),good water-solubility (at approx. neutral pH) and low toxicity. Toxicityis less of a concern for topical administration, as is water solubility.

4.2.5 Compositions and Uses

The antimicrobial sulfonamide derivatives of the invention can be usedin a wide variety of applications to inhibit the growth or killmicroorganisms. For example, the antimicrobial sulfonamide derivativesmay be used as disinfectants or as preservatives for materials such asfoodstuffs, cosmetics, medicaments and other nutrient containingmaterials. The antimicrobial sulfonamide derivatives can also be used totreat or prevent diseases related to microbial infection in subjectssuch as plants and animals.

For use as a disinfectant or preservative, the antimicrobial sulfonamidederivatives can be added to the desired material singly, as mixtures ofantimicrobial sulfonamide derivatives or in combination with otherantifungal and/or antimicrobial agents. The antimicrobial sulfonamidederivatives may be supplied as the compound per se or may be inadmixture with a variety of adjuvants, carriers, diluents or excipients,which are well known in the art.

When used to treat or prevent microbial infections or diseases relatedthereto, the antimicrobial sulfonamide derivatives of the invention canbe administered or applied singly, as mixtures of two or moreantimicrobial sulfonamide derivatives, in combination with otherantifungal, antibiotic or antimicrobial agents or in combination withother pharmaceutically active agents. The antimicrobial sulfonamidederivatives can be administered or applied per se or as pharmaceuticalcompositions. The specific pharmaceutical formulation will depend uponthe desired mode of administration, and will be apparent to those havingskill in the art. Numerous compositions for the topical or systemicadministration of antibiotics are described in the literature. Any ofthese compositions may be formulated with the antimicrobial sulfonamidederivatives of the invention.

Pharmaceutical compositions comprising the antimicrobial sulfonamidederivatives of the invention may be manufactured by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.Pharmaceutical compositions may be formulated in conventional mannerusing one or more physiologically acceptable adjuvants, carriers,diluents, excipients or auxiliaries which facilitate processing of theactive antimicrobial sulfonamide derivatives into preparations which canbe used pharmaceutically. Proper formulation is dependent upon the routeof administration chosen.

For topical administration the antimicrobial sulfonamide derivatives ofthe invention may be formulated as solutions, gels, ointments, creams,suspensions, etc. as are well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration.

For injection, the antimicrobial sulfonamide derivatives of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer. The solution may containformulatory agents such as suspending, stabilizing and/or dispersingagents.

Alternatively, the antimicrobial sulfonamide derivatives may be inpowder form for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the antimicrobial sulfonamide derivatives canbe readily formulated by combining them with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. For oral solid formulations suchas, for example, powders, capsules and tablets, suitable excipientsinclude fillers such as sugars, such as lactose, sucrose, mannitol andsorbitol; cellulose preparations such as maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP); granulating agents; and binding agents. Ifdesired, disintegrating agents may be added, such as the cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate. If desired, solid dosage forms may be sugar-coated orenteric-coated using standard techniques.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added.

For buccal administration, the compositions may take the form oftablets, lozenges, etc. formulated in conventional manner.

For administration by inhalation, the compounds of the present inventionare conveniently delivered in the form of an aerosol spray frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The antimicrobial sulfonamide derivatives may also be formulated inrectal or vaginal compositions such as suppositories or retentionenemas, e.g., containing conventional suppository bases such as cocoabutter or other glycerides.

In addition to the formulations described previously, the antimicrobialsulfonamide derivatives may also be formulated as a depot preparation.Such long acting formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the compounds may be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well known examples of delivery vehiclesthat may be used to deliver the antimicrobial sulfonamide derivatives ofthe invention. Certain organic solvents such as dimethylsulfoxide alsomay be employed, although usually at the cost of greater toxicity.Additionally, the antimicrobial sulfonamide derivatives may be deliveredusing a sustained-release system, such as semipermeable matrices ofsolid polymers containing the therapeutic agent. Varioussustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the compounds for a few weeks up to over100 days.

As certain of the carboxylic acids of the antimicrobial sulfonamidederivatives of the invention are acidic, or the lipophilic group orlinker may include acidic or basic substituents, the antimicrobialsulfonamide derivatives may be included in any of the above-describedformulations as the free acids, the free bases or as pharmaceuticallyacceptable salts.

The antimicrobial sulfonamide derivatives of the invention, orcompositions thereof, will generally be used in an amount effective toachieve the intended purpose. Of course, it is to be understood that theamount used will depend on the particular application.

For example, for use as a disinfectant or preservative, anantimicrobially effective amount of a antimicrobial sulfonamidederivative, or composition thereof, is applied or added to the materialto be disinfected or preserved. By antimicrobially effective amount ismeant an amount of antimicrobial sulfonamide derivative or compositionthat inhibits the growth of, or is lethal to, a target microbe. Whilethe actual amount will depend on a particular target microbe andapplication, for use as a disinfectant or preservative the antimicrobialsulfonamide derivatives, or compositions thereof, are usually added orapplied to the material to be disinfected or preserved in relatively lowamounts. Typically, the antimicrobial sulfonamide derivatives comprisesless than about 5% by weight of the disinfectant solution or material tobe preserved, preferably less than about 1% by weight and morepreferably less than about 0.1% by weight. An ordinarily skilled artisanwill be able to determine antimicrobially effective amounts ofparticular antimicrobial sulfonamide derivatives for particularapplications without undue experimentation using, for example, the invitro assays provided in the examples.

For use to treat or prevent microbial infections, the antimicrobialsulfonamide derivatives of the invention, or compositions thereof, areadministered or applied in a therapeutically effective amount. Bytherapeutically effective amount is meant an amount effective toameliorate the symptoms of, or ameliorate, treat or prevent microbialinfections. Determination of a therapeutically effective amount is wellwithin the capabilities of those skilled in the art, especially in lightof the detailed disclosure provided herein. Preferably, atherapeutically effective amount is between about 20 mg/kg and about 0.5mg/kg, more preferably between about 10 mg/kg and about 1 mg/kg, mostpreferably between about 5 mg/kg and about 2 mg/kg.

As in the case of disinfectants and preservatives, a therapeuticallyeffective dose, for topical administration to treat or preventmicrobial, yeast, fungal or other infection, can be determined usingconventional methods by the skilled artisan. The treatment may beapplied while the infection is visible, or even when it is not visible.An ordinarily skilled artisan will be able to determine therapeuticallyeffective amounts to treat topical infections without undueexperimentation.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating antimicrobialsulfonamide derivative concentration range that includes the IC₅₀ asdetermined in cell culture (i.e., the concentration of test compoundthat is lethal to 50% of a cell culture), the MIC as determined in cellculture (i.e., the minimal inhibitory concentration for growth) or theIC₁₀₀ as determined in cell culture (i.e., the concentration ofantimicrobial sulfonamide derivative that is lethal to 100% of a cellculture). Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data (e.g., animalmodels) using techniques that are well known in the art. One of ordinaryskill in the art can readily optimize administration to humans based onanimal data.

Alternatively, initial dosages can be determined from the dosagesadministered of known antimicrobial agents (e.g., aspartocin,laspartomycin etc.) by comparing the IC₅₀, MIC and/or I₁₀₀ of thespecific antimicrobial sulfonamide derivatives with that of a knownantimicrobial agent, and adjusting the initial dosages accordingly. Theoptimal dosage may be obtained from these initial values by routineoptimization.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active antimicrobial sulfonamide derivatives whichare sufficient to maintain therapeutic effect. Usual patient dosages foradministration by injection range from about 0.1 to 5 mg/kg/day,preferably from about 0.5 to 1 mg/kg/day. Therapeutically effectiveserum levels may be achieved by administering a single daily dose ormultiple doses each day.

In cases of local administration or selective uptake, the effectivelocal concentration of antimicrobial sulfonamide derivative may not berelated to plasma concentration. One having skill in the art will beable to optimize therapeutically effective local dosages without undueexperimentation.

The amount of antimicrobial sulfonamide derivative administered will, ofcourse, be dependent on, among other factors, the subject being treated,the subject's weight, the severity of the affliction, the manner ofadministration and the judgment of the prescribing physician.

The antimicrobial therapy may be repeated intermittently whileinfections are detectable, or even when they are not detectable. Thetherapy may be provided alone or in combination with other drugs, suchas for example other antibiotics or antimicrobials, or otherantimicrobial sulfonamide derivatives of the invention.

Preferably, a therapeutically effective dose of the antimicrobialsulfonamide derivatives described herein will provide therapeuticbenefit without causing substantial toxicity. Toxicity of theantimicrobial sulfonamide derivatives can be determined using standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index.Antimicrobial sulfonamide derivatives which exhibit high therapeuticindices are preferred. The data obtained from these cell culture assaysand animal studies can be used in formulating a dosage range that is nottoxic for use in subjects. The dosage of the antimicrobial sulfonamidederivatives described herein lies preferably within a range ofcirculating concentrations that include the effective dose with littleor no toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition (See, e.g.,Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.1,p.1).

5. EXAMPLES

The invention having been described, the following examples arepresented to illustrate, rather than limit, the scope of the invention.The examples illustrate various embodiments and features of the presentinvention.

5.1 Preparation of Deacylase Enzyme

The deacylase is produced by culturing Actinoplanes utahensis NRRL 12052under submerged aerobic fermentation conditions. Because single-colonyisolates from a lyophile of the culture were heterogeneous for bothmorphology and enzyme production capability, selections were made torecover a stable, high-producing variant. Initially, multiplefermentations were carried out using inocula prepared from strain 12052.Vegetative growth from the flask yielding high deacylating activity wasplated on a differential agar (CM). Colonies were then selected forfurther evaluation. Generally, small colony types were better enzymeproducers than large colony types. Isolate No. 18 was the best deacylaseproducer of all selected colonies and was routinely used for theproduction of the deacylase enzyme. CM agar contained corn steep liquor0.5%, Bacto peptone 0.5%, soluble starch 1.0%. NaCl 0.05%, CaCl₂.2H₂O0.05% and Bacto agar 2.0%.

The high-producing, natural variant was used in the known fermentationprotocol employed (Boeck et al., 1988. J. Antibiot., 41, 1085). A stockculture of Actinoplanes utahensis NRRL 12052 variant, preserved in 20%glycerol at −70° C., was introduced into a 25×150 mm test tubecontaining 10 mL of a medium comprised of 2.0% sucrose, 2.0% pre-cookedoatmeal, 0.5% distiller's grain, 0.25% yeast, 0.1% K₂HPO₄, 0.05% KCl,0.05% MgSO₄-7H₂O and 0.0002% FeSO₄-7H₂O in deionized water. Afterincubation at 30° C. for 72 hrs on a rotary shaker orbiting at 250 rpmthe resulting mycelial suspension was transferred into 50 mL of PM3medium in a 250 mL Erlenmeyer flask. The PM3 medium contained 2.0%sucrose, 1.0% peanut meal, 0.12% K₂HPO₄, 0.05% KH₂PO₄ and 0.025%MgSO₄-7H₂O, in tap water. The flask was incubated at a temperature of30° C. for a period of between 60 to 90 hrs. The whole broth from thisfermentation contained the deacylase enzyme.

5.2 Synthesis of Decanesulfonylglycyl Aspartocin

5.2.1 Extractive Purification of Aspartocin

Approximately 20 grams of a crude preparation of aspartocin (see e.g.,Shay et al., 1960, Antibiotics Annual, 194) was mixed with about 125 mLof water and insoluble impurities were separated by centrifugation.About 300 mg of CaCl₂ was added to the brown colored liquid and theresulting solution was adjusted to a pH of between about 8.6 to about pH9.0. Aspartocin was then extracted into about 100 mL of 1-butanol. Theaqueous phase was again mixed with about 600 mg of CaCl₂ and thenextracted with 1-butanol. The combined butanol extracts were mixed withan equal amount of water, the mixture adjusted to about pH 2.0 and thebutanol phase washed with about 160 mL of water adjusted toapproximately pH 2.0. Aspartocin, was then extracted into water at aboutpH 7.0 and then back into butanol at a pH of between about pH 2.0 toabout pH 3.0. The butanol phase was washed with about 100 mL of water atapproximately pH. 2.0, then combined with an equal volume of water andadjusted to about pH 7.0. The pH adjustments were made with 1 N HCl and1 N NaOH. The aqueous phase is evaporated under vacuum to removeresidual butanol. The very slightly colored clear liquid wasfreeze-dried to obtain 803 mg of the sodium salt of aspartocin astan-white powder. FAB-MS m/z:1340 (M+Na)⁺, 1362 (M+2Na−H)⁺, 1384(M+3Na−2H)⁺, 1406 (M+4Na−3H)⁺.

5.2.2 Preparation of FMOC-Aspartocin

Aspartocin (0.54 g, 0.41 mmol) preferably purified as described inSection 5.2.1, and 0.30 mL (1.73 mmol) of diisopropylethylamine weredissolved in 5 mL of H₂O, cooled in an ice bath.9-fluorenylmethyloxycarbonyl (“FMOC”) chloride, 0.26 g (1.0 mmol) wasdissolved in 2.0 mL of dioxane and 1.0 mL of this solution added to thecooled aspartocin solution. High Pressure Liquid Chromatography (“HPLC”)analysis indicated that the reaction was almost complete after 0.5 hour,at which time the remaining 1.0 mL of FMOC chloride solution was addedto the reaction mixture. After 15 minutes 6 mL of H₂O was added and thereaction mixture was extracted twice with ethyl acetate (“EtOAc”).

The aqueous phase was filtered through Celite, evaporated to removeresidual EtOAc, and freeze-dried to obtain 0.56 g of product. The saltwas then dissolved in 15 mL of H₂O, centrifuged to remove a small amountof insoluble material, acidified to pH 1.88 with 1 N HCl, separated fromthe acidified solution by centrifugation, washed with H₂O, and dried invacuo over P₂O₅ to yield 0.31 g of the free acid. FAB-MS of the majorcomponent had m/z 1542 (M+H)⁺.

5.2.3 Deacylation of FMOC-Aspartocin

FMOC-aspartocin (476 mg of about 65% purity) prepared as described inSection 5.2.2 was dissolved in 30 mL of 0.5 M potassium phosphatebuffer, pH 7.1, to which 300 mL of deacylase fermentation broth wasadded. The reaction mixture was incubated for about 18 hours at about29° C. Conversion of FMOC-Aspartocin to the deacylated product wasestimated to be about 72% complete by HPLC analysis. Acetonitrile (150mL) was added to the fermentation broth, the mixture sonicated for about1 minute, centrifuged at 3000 rpm for 5 minutes and decanted. Thedecanted supernatant was diluted with an equal volume of distilled water(total volume about 900 mL), split into three equal portions, andapplied to three styrene-divinylbenzene resin cartridges (ENVI-Chrom Presin, 3.1 g per cartridge, 25×30 mm). The cartridges were eluted bygravity flow using a stepwise gradient in acetonitrile buffered with0.025 M potassium phosphate at pH 7.1. The deacylated product was elutedwith a 23-27% acetonitrile gradient. Appropriate fractions were pooledand acetonitrile removed under vacuum at room temperature. The threeresin cartridges were regenerated by elution with acetonitrile, followedby equilibration with 5% acetonitrile in unbuffered water and the pooledfractions were then applied to the cartridges. Excess salt was removedby washing with 5% acetonitrile, while the product was eluted with 50%acetonitrile. Acetonitrile was removed from the combined desaltedproduct fractions under vacuum at room temperature and the fractionswere freeze dried to yield 184 mg of an orange-tan solid, C₆₀H₇₉N₁₃O₂₁of deacylated FMOC-aspartocin (i.e., the FMOC derivative of the aminocore antibiotic of aspartocin). FAB-MS: m/z 1356(M+K)⁺, 1394(M−H+2K)⁺.Calculated for C₆₀H₇₉N₁₃O₂₁+K, 1356).

5.2.4 Sulfonation of Deacylated FMOC-Aspartocin

About 13.5 mg of deacylated FMOC-aspartocin (prepared as described inSection 5.2.3; approximately 65% pure as the major component) wasdissolved in about 1.0 mL of dimethylformamide containing 0.01 mL ofdiisopropylethylamine. The hydroxybenzotriazole activated ester ofdecanesulfonylglycine was added incrementally to the above solution atroom temperature and the reaction monitored by HPLC. After about 2.5hours the conversion to product was estimated to be about 71% with thetotal amount of presumed product about 6 mg. The reaction mixture waspoured over 4.0 g ice and the pH adjusted to about 6.7 by addition of 2drops of H₃PO₄, 2 mL of 0.5M ammonium phosphate buffer pH 7.2 and 2 mLof acetonitrile. After storage in the freezer for about 3 days thethawed reaction mixture was diluted with 2 mL of 0.5 M acetate buffer(pH 4.6) and 4 mL of distilled water; the pH was adjusted to about pH4.9 by addition of acetic acid to provide a slightly turbid solution.The solution was applied to a divinylbenzene-styrene resin cartridge(Supelco ENVI-Chrom P, 5.0 g, 25×40 mm) with gravity flow. The cartridgewas eluted with a stepwise gradient of pH 4.6 acetate-buffered aqueousacetonitrile with the product being eluted with 45% acetonitrile.Fractions were pooled, acetonitrile removed under vacuum at roomtemperature and the resulting aqueous solution freeze dried to yield 5.5mg of white solid. FAB-MS: m/z 1617(M+K)⁺. Calculated forC₇₂H₁₀₂N₁₄O₂₄S+K, 1617.

5.2.5 Deprotection of Decanesulfonylglycyl-FMOC-Aspartocin

One drop of piperdine was added to about 5.5 mg of the product fromSection 5.2.4 dissolved in 2.0 mL of 2:1 mixture ofdimethylsulfoxide:methanol. Monitoring by HPLC indicated the reactionwas complete after 60 minutes at room temperature. The reaction wasquenched by addition of 6.0 mL cooled 0.2 M ammonium phosphate at pH7.2. The quenched reaction mixture was applied to a 0.5 g resincartridge (ENVI-Chrom P) and eluted with a stepwise gradient of pH 7.2phosphate-buffered aqueous acetonitrile. The product was eluted with 25%acetonitrile. Appropriate fractions were pooled and acetonitrile wasremoved under vacuum at room temperature. The product was desalted byadsorption onto a 0.5 g resin cartridge which was then rinsed with 10%acetonitrile, followed by 50% acetonitrile. The 50% acetonitrile eluatewas evaporated to remove acetonitrile and the resulting aqueous solutionfreeze dried to provide about 1.5 mg of white solid. FAB-MS: m/z 1379(M+Na)⁺ was consistent with the expected structure. The MIC values forthe decanesulfonylglycyl derivative against Staphylococcus aureus wereessentially the same as those for aspartocin.

5.2.6 Scale-Up of Sulfonation of Deacylated FMOC-Aspartocin

About 68 mg of deacylated FMOC-aspartocin (prepared as described inSection 5.2.3; approximately 65% pure as the major component) wasdissolved in about 1.0 mL dimethylformamide. The HOBT activated ester ofdecanesulfonylglycine was added incrementally to the peptide solutionand the reaction was monitored by HPLC until the conversion to productwas about 95%. The total amount of presumed product was estimated to beabout 32.0 mg. The reaction mixture was diluted with about 5 mL ofmethanol, the apparent pH adjusted to between about 6-7 using 1.5M NH₄OHand then filtered through a 0.45 μm membrane. The filtrate waschromatographed on a Sephadex LH-20 size exclusion column, which waseluted with methanol. Fractions containing product were pooled andmethanol was evaporated under vacuum to provide about 64 mg of solidresidue, which contained about 29 mg of product. The partially purifiedproduct was deblocked by dissolving in about 2 mL ofdimethylsulfoxide/methanol (2/1), adding 2 drops of piperdine andstirring at about 25° C. for 75 minutes. The reaction mixture was thendiluted with about 20 mL of 10% acetonitrile buffered ammonium phosphate(apparent pH of about 7.8), and then filtered through a 0.45 μmmembrane. The filtrate was applied to a 2.5×8.5 cmstyrene-divinylbenzene resin column (Supelco ENVI-Chrom P, 9.3 g) andeluted with increasing concentrations of acetonitrile in aqueousammonium phosphate at pH7.2. The presumed product was eluted in 30%acetonitrile. Appropriate fractions were pooled, the acetonitrile wasevaporated under vacuum, and the solution was desalted and freeze driedin similar fashion as described in Section 5.2.4. Yield: 2 mg product ofca. 80% purity. Additional product was recovered by pooling sidefractions from the resin column isolation and rechromatographing on a5.0 g resin column; product was eluted with ca. 28% acetonitrile.Appropriate fractions were pooled and the desalted product was recoveredin similar fashion as described above. The first 21 mg of product wasadded to the desalted rechromatographed side fractions pool and freezedried. Overall yield: 29 mg of white solid of 80% purity based on HPLC,C₅₇H₉₂N₁₄O₂₂S, FABMS: m/z 1357, (M+H)⁺, 1379(M+Na)⁺, 1395(M+K)⁺.

5.3 Synthesis of the Amino Core Antibiotic and Amino Core Cyclic Peptideof Laspartomycin

5.3.1 Biochemical Synthesis of the Amino Core Cyclic Peptide ofLaspartomycin

Laspartomycin (257 mg) in about 12 mL of 0.5M phosphate buffer of aboutpH 7.2 was added to about 120 mL of deacylase fermentation brothprepared as in Section 5.1 and incubated for about 16 hours at about 29°C. at about 180 rpm. The broth was centrifuged, the centrifugatedecanted and solids were extracted with about 40 mL of distilled water.The pooled centrifugates were then applied to a 2.5×5.0 cmstyrene-divinylbenzene resin column (ENVI™-Chrom P) and the product waseluted with a 10% and 11% acetonitrile-pH 7.2 phosphate mixture. Pooledfractions were concentrated and the pH was adjusted to about 4.65 byaddition of ammonium acetate-acetic acid buffer. The fractions were thenapplied to a 2.5×5.0 cm resin column (ENVI™-Chrom P). The desiredmaterial was eluted with a 12.5% acetonitrile-ph 4.65 acetate mixture.The pH of the pooled fractions was adjusted to about 7.8, followed byconcentration and freeze-dried to provide about 74 mg of the amino corecyclic peptide of laspartomycin as an off-white solid which was about97% pure when analyzed by High Pressure Liquid Chromatography (“HPLC”)at 215 nm. FAB-MS m/z 910 (HR-FAB-MS of the amino core cyclic peptide oflaspartomycin: found 910.4251 (M+H)⁺, calc. 910.4270 forC₃₈H₅₉N₁₁O₁₅+H). Also obtained was about 14 mg of an isomer of the aminocore cyclic peptide of laspartomycin as an off white solid. FAB-MS: m/z910(M+H)⁺.

5.3.2 Biochemical Synthesis of the Amino Core Antibiotic and Amino CoreCyclic Peptide of Laspartomycin

About 2.5 g of laspartomycin was treated with the deacylase broth underconditions similar to those described in Example 5.3.1 except whereexplicitly noted. About 1.0 g of laspartomycin was treated withdeacylase fermentation broth at about 2.0 mg/mL for about 3.7 hrs toproduce a sample enriched in the amino core antibiotic of laspartomycin.About 1.5 g of laspartomycin was treated with deacylase fermentationbroth at about 5.0 mg/mL for about 20 hours. The fermentation brothswere pooled and then processed as described in 5.3.1 to provide about100 mg of the amino core antibiotic of laspartomycin and about 600 mg ofthe amino core cyclic peptide of laspartomycin, and an estimated 150 mgof an isomer of the amino core cyclic peptide of laspartomycin. FAB-MSof the amino core antibiotic of laspartomycin: m/z 1026(M+H)⁺,1048(M+Na)⁺.

5.3.3 Synthesis of Protected Amino Core Antibiotic of Laspartomycin

Equimolar amounts of t-butoxycarbonyl-L-aspartic acid 4-O-t-butyl ester,dicyclohexylcarbodiimide, and 1-hydroxybenzotriazole in tetrahydrofuranwas stirred overnight and the reaction mixture was filtered andevaporated to give a crystalline solid. The solid was then slurried inethyl acetate, filtered and dried to provide t-butoxycarbonyl-L-asparticacid-4-O-t-butyl ester 1-hydroxybenzotriazole ester.

A mixture of the amino core cyclic peptide of laspartomycin (15.2 mg,0.0167 mmol) and diisopropylethlyamine (0.025 mL, 0.1437 mmol) in 0.20mL of dimethylformamide was stirred at room temperature under nitrogen.A solution of t-butoxycarbonyl-L-aspartic acid-4-O-t-butyl ester1-hydroxybenzotriazole ester (0.030 mL aliquots) containing 0.0496 mg(0.1218 mmol) of the activated ester in 0.20 mL was initially added andagain after 0.50 hr. The progress of the reaction was followed by HPLC.Water was added to quench the reaction and the reaction mixture adsorbedon a 2.5×5.0 cm styrene-divinylbenzene resin column (ENVI™-Chrom P), andeluted with pH 7.2 phosphate buffer containing about 45% acetonitrile.Fractions containing the desired product were desalted and freeze driedto obtain 9.0 mg of the protected amino core antibiotic of laspartomycinestimated 90% pure based on HPLC. FAB-MS: m/z 1182 (M+H)⁺, 1204 (M+Na)⁺.

5.3.4 Synthesis of the Amino Core Antibiotic of Laspartomycin

0.35 mL of trifluoroacetic acid was added to 6.9 mg of the compoundprepared above and the solution was allowed to stand at room temperaturefor 1.5 hr. Trifluoroacetic acid was removed and the residue waslyophilized to afford 4.8 mg of the amino core antibiotic oflaspartomycin as the trifluoroacetate salt. FAB-MS: m/z 1025 (M+H)⁺,1047 (M+Na)⁺, 1063 (M+K)⁺.

5.4 Synthesis of the Hexadecylsulfonyl-L-Tryptophan Derivative of theCore Antibiotic of Laspartomycin

5.4.1 Hexadecylsulfonyl-L-Tryptophan Methyl Ester

A solution of hexadecylsulfonyl chloride (260 mg, 0.80 mmol), tryptophanmethyl ester hydrochloride (254 mg, 1.0 mmol), and 0.34 mL oftriethylamine (2.45 mmol) in 2.0 mL of dimethylformamide was stirred atroom temperature for 4.0 hrs. The mixture was diluted with 10 mL of 1.0N HCl and extracted with 20 mL of ethyl acetate. The ethyl acetatesolution was washed with water and saturated salt solution and driedover magnesium sulfate then evaporated to give beige crystals. Yield 261mg, FABMS: m/z 507 (M+H)⁺.

5.4.2 Hexadecylsulfonyl-L-Tryptophan

A mixture of hexadecylsulfonyl-L-tryptophan methyl ester (260 mg 0.514mmol), and 0.50 mL of 1.0 N NaOH in 2.0 mL of methanol and 2.0 mL oftetrahydrofuran was stirred at room temperature for several hrs. Thinlayer chromatography indicated the reaction was incomplete. Anadditional 0.50 mL of 1.0 N NaOH was added and the mixture was stirreduntil reaction was complete (about 18 hrs.). The reaction was worked upas described above to afford 196 mg of product. FABMS: m/z 493 (M+H)⁺.

5.4.3 Hexadecylsulfonyl-L-Tryptophan Derivative of the Core Antibioticof Laspartomycin

Hexadecylsulfonyl-L-Tryptophan (90 mg, 0183 mmol), hydroxybenzotriazole(28 mg, 0.183 mmol), and dicyclohexylcarbodiimide (38 mg, 0.183 mmol)was stirred for 40 minutes in 1.0 mL of dimethylformamide. A 0.30 mLaliquot of this solution was added to a solution of the amino coreantibiotic of laspartomycin (94.5 mg, 0.0439 mmol) in 0.20 mL ofdimethylformamide. The progress of the reaction was monitored by HPLC.At the completion of the reaction, the reaction mixture was diluted with5 mL of methanol and 1.5M NH₄OH was added to an apparent pH of about 7.The filtered sample solution was applied to a 2.5×44 cm size exclusioncolumn (Sephadex LH-20 fine, swelled in methanol) which was eluted withmethanol at about 0.8 mL/min. The product eluted in about 25 mL ofeluate starting at about 105 mL. The methanol was removed from theproduct pool by evaporation under vacuum at or below 30° C. The solidresidue was dissolved in about 12 mL of 10% acetonitrile buffered with0.08M ammonium phosphate (aqueous pH 7.2). This solution was applied toa 2.5×5 cm styrene-divinylbenzene resin column (Supelco ENVI-Chrom Presin) and eluted with increasing concentrations of acetonitrilebuffered with pH7.2 ammonium phosphate. The product eluted in about 36mL using 48% acetonitrile eluent. Acetonitrile was removed from thisfraction by evaporation under vacuum. Prior to applying this fraction tothe resin column for desalting, the resin column was washed with 70%acetonitrile, then 100% acetonitrile, and finally 20% acetonitrile. Theaqueous solution of the sample was applied then to the column. Thecolumn was rinsed with 28 mL of 21% acetonitrile (unbuffered) and thedesalted product was stripped from the column using 67% acetonitrile.Acetonitrile was removed by evaporation under vacuum and the product wasfreeze dried. Yield: 14 mg white solid, C₆₉H₁₀₄N₁₄O₂₁; FABMS: m/z 1499(M+H)⁺, 1521 (M+Na)⁺, 1537(M+K)⁺.

5.5 Synthesis of the Hexadecylsulfonyl-L-Phenylalanine Derivative of theCore Antibiotic of Laspartomycin

5.5.1 Hexadecylsulfonyl-L-Phenylalanine Methyl Ester

A solution of hexadecylsulfonyl chloride (617 mg, 1.87 mmol),L-phenylalanine methyl ester hydrochloride (501 mg, 2.32 mmol), and 0.60mL of triethylamine (4.33 mmol) in 4.0 mL of dimethylformamide wasstirred at room temperature for 4.0 hrs. The mixture was diluted with 20mL of 1.0 N HCl and extracted with 20 mL of ethyl acetate. The ethylacetate solution was washed with water and saturated salt solution anddried over magnesium sulfate then evaporated to give an oil whichcrystallized on standing; yield 595 mg, FABMS: m/z 468 (M+H)⁺.

5.5.2 Hexadecylsulfonyl-L-Phenylalanine

A mixture of hexadecylsulfonyl-L-phenylalanine methyl ester (590 mg,1.26 mmol), and 2.0 mL of 1.0 N NaOH in 4.0 mL of methanol and 2.0 mL oftetrahydrofuran was stirred at room temperature until the reaction wascomplete as shown by Thin Layer Chromatography. Work up was as describedfor the corresponding tryptophan analog in 5.4.2 above and gave thedesired product; FABMS: 454 (M+H)⁺476 (M+N)⁺.

5.5.3 Hexadecylsulfonyl-L-Phenylalanine Derivative of the CoreAntibiotic of Laspartomycin

Hexadecylsulfonyl-L-phenylalanine (60 mg, 0.132 mmol),hydroxybenzotriazole (19 mg, 0.132 mmol), and dicyclohexylcarbodiimide(32 mg, 0.151 mmol) was stirred for 40 minutes in 0.67 mL ofdimethylformamide. A 0.30 mL aliquot of this solution was added to asolution of the amino core antibiotic of laspartomycin (50 mg., 0.0488mmol) in 0.40 mL of dimethylformamide. The progress of the reaction wasmonitored by HPLC and the product was isolated by chromatography in asimilar fashion to that described in 5.4.3. Product was a white powder,13 mg, C₆₇H₁₀₅N₁₃O₂₁S, FABMS: m/z 1460.5 (M+H)⁺, 1482.4 (M+Na)⁺, 1498.4(M+K)⁺.

5.6 Synthesis of the Hexadecylsulfonyl Derivative of the Core Antibioticof Laspartomycin

5.6.1 N-Hexadecylsulfonyl Derivative of the (O-t-butyl) Core Antibioticof Laspartomycin

N-hexadecylsulfonyl-(O-t-butyl)-L-aspartic acid (189 mg, 0.395 mmol,1-hydroxybenzotriazole (55 mg, 0.395 mmol) and dicyclohexylcarbodiimide(83 mg, 0.395 mmol) in 0.50 mL of dimethylformamide was stirred at roomtemperature for forty five minutes. A 0.050 mL aliquot of this solutionwas added to the tetrabutylammonium salt of the amino core cyclicpeptide of laspartomycin in 0.2 mL of dimethylformamide and stirred atroom temperature for sixty minutes. The reaction mixture was quenched bydilution with 8 mL of 25% acetonitrile, 0.12 M in ammonium phosphate (pH7.2), aged at room temperature, then membrane filtered (Whatman GD/X).The product was isolated from the filtrate by low resolution reversephase chromatography on a 5 g styrene-divinylbenzene resin cartridge(25×45 mm, Supelco EnviChrom-P). The sample-loaded cartridge was elutedwith stepwise increasing concentrations of acetonitrile in sodiumphosphate (aqueous pH 6.9); the product was eluted with 57%acetonitrile, 0.010 M in pH 6.9 buffer. The material was then desaltedas described in Section 5.4.3. Yield: 4.7 mg of white solid, 69% by HPLC(215 nm area %); C₆₂H₁₀₄N₁₂O₂₀S.

5.6.2 N-Hexadecylsulfonyl Derivative of the Core Antibiotic ofLaspartomycin

A solution of N-hexadecylsulfonyl-(O-t-butyl)-L-aspartyl-laspartomycincore antibiotic (4.7 mg) in 0.50 mL of 95% trifluoroacetic acid wasstirred at room temperature for 30 min. Trifluoroacetic acid was removedwith a stream of dry nitrogen and the residue was triturated witht-butylmethyl ether and centrifuged. Excess ether was removed and theresulting solid was dissolved in 1.5 mL of water by adding 1 drop of 3%ammonium hydroxide, then freeze dried. Yield: 2.5 mg of solid, 63% byHPLC (215 nm area %); C₅₈H₉₆N₁₂O₂₀S; FABMS m/z 1313 (M+H)⁺, 1335(M+Na)⁺. Calculated for C₅₈H₉₆N₁₂O₂₀S+H, 1313.

5.7 Synthesis of the Hexadecylsulfonyl-L-Phenylalanine Derivative of theCore Antibiotic of Aspartocin

5.7.1 N-Hexadecylsulfonyl-L-Phenylalanine Derivative of the CoreAntibiotic of FMOC-Aspartocin

A mixture of N-hexadecylsulfonyl-L-phenylalanine (100 mg, 0.220 mmol),1-hydroxybenzotriazole (35.5 mg, 0.232 mmol), anddicyclohexylcarbodiimide (45 mg, 0.218 mmol) in 0.95 mL ofdimethylformamide was stirred at room temperature for 45 minutes. A 0.30mL aliquot of this solution was added to a solution of the FMOCderivative of the amino core antibiotic of aspartocin (80 mg, obtainedfrom FMOC-Aspartocin as described in section 5.2.3) in 0.50 mLdimethylformamide. Additional aliquots of 0.30 mL and 0.15 mL of theactivated ester were added after 35 and 70 minutes. The reaction mixturewas quenched by addition of 5 mL of methanol and the pH was adjusted topH 7 (using pH paper) by addition of 0.55 mL of 1.5 M ammoniumhydroxide. The quenched solution was membrane filtered (Whatman GD/X)and the filtrate was applied to a Sephadex LH-20 column (25×420 mm)equilibrated in methanol. The sample was eluted with methanol at about0.8 mL/min and the methanol was removed under vacuum. The product wasfurther purified by preparative HPLC (Waters Delta-Pak C 18 column,25×110 mm) using 53% isopropanol and 0.03 M in ammonium acetate (aqueouspH 5.4) at a flow rate of 10 mL/min at room temperature. Isopropanol wasremoved under vacuum and the pH of the turbid aqueous solution wasadjusted to pH 6 with dilute ammonium hydroxide. The product wasdesalted and freeze dried as previously described above. Yield: 16 mg ofa white solid, 66% by HPLC (215 nm area %); C₈₅H₁₂₀N₁₄O₂₄S; FABMS m/z1776 (M+Na)⁺, 1791(M+K)⁺. Calculated for C₈₅H₁₂₀N₁₄O₂₄S+Na, 1776).

5.7.2 N-Hexadecylsulfonyl-L-Phenylalanine Derivative of the CoreAntibiotic of FMOC-Aspartocin

A solution of 10.4 mg of the product of Section 5.7.1 in 1.0 mL of 2/1DMSO-methanol and 0.020 mL of piperdine was stirred for 90 minutes atroom temperature, diluted with 10 mL of 20% acetonitrile in 0.040 M inammonium phosphate (pH7.2), then membrane filtered (Whatman GD/X). Thefiltrate was applied to a conditioned 0.5 g resin cartridge (SupelcoEnviChrom-P) which was then rinsed with salt-free 20% acetonitrile (6mL). The product was stripped from the cartridge with 4 mL of 60%acetonitrile, acetonitrile was removed under vacuum and the productfreeze dried from aqueous solution. Yield: 7 mg of a white solid, 74% byHPLC (215 nm area %); C₇₀H₁₁₀N₁₄O₂₂S; FABMS m/z 1533(M+H)⁺, 1555(M+Na)⁺,1571(M+K)⁺. Calculated for C₇₀H₁₁₀N₁₄O₂₂S+Na, 1532, (instrument error atthis mass range is ±0.5 mass units).

5.8 Resistance of Sulfonamide Derivatives to the Deacylase Enzyme

One milligram of the hexadecylsulfonyl-L-tryptophan derivative of thecore antibiotic of laspartomycin (see Section 5.4 for preparation) wasadded to 2 mL of deacylase broth (prepared as described in Section 5.1)and a zero time sample (0.5 mL) was removed and stored by freezing. Thereaction mixture was placed on a shaker at 200 rpm and 84° C. for 16hours. The zero and 16 hour samples were then studied by HPLC and byagar well zone diffusion assay using Staphylococuss aureus as a testorganism. The same conditions were used to compare laspartomycin toenzyme deactivation. The table below shows the results which indicatecomplete deactivation of laspartomycin by the enzyme preparation whilethe hexadecylsulfonyl-L-tryptophan derivative of the core antibiotic oflaspartomycin was degraded at a much slower rate. Under the sameconditions Antibiotic A21978C and Aspartocin were completely deactivatedwithin two hours and 16 hours, respectively.

Bioassay zone dia.^(a) HPLC Peak Area^(b) Compound Zero time 16 hourszero time 16 hours Laspartomycin 15.5 mm no zone 100%  0%Hexadecylsulfonyl-L- 13.7 mm 12.8 mm 100% 53% tryptophan derivative ofthe core antibiotic of laspartomycin ^(a)9 mm well in agar seeded withStaphylococuss aureus wells filled with 100 μl of sample and incubatedfor 16 hrs. at 28° C. ^(b)HPLC with reverse phase column, Phenomenex, ®Prodigy ™ 5 μ ODS(2), 250 × 4.60 mm, using gradient elution; gradientwas 0.05 molar phosphate buffer, pH 7.2, with 10% CH₃CN going to 75%CH₃CN in water in 8 minutes. Retention times were 9.62(main component)and 13.13 minutes for laspartomycin and thehexadecylsulfonyl-L-tryptophan derivative of the core antibiotic oflaspartomycin, respectively.

5.9 MIC Data for Antimicrobial Sulfonamides

MIC values were determined by microliter serial dilution usingStaphlococcus aureus strain Smith as the assay organism, which was grownin Mueller-Hinton broth with and without CaCl₂.

MIC(mg/mL) Name w/o CaCl₂ w CaCl₂ (4 mm) Aspartocin 2 1 Laspartomycin16  2 52 8-16 4 54 >64  >64  56 8 4 58 32  1 60 >64  >64  62 4 4

While the invention has been described in some detail to facilitateunderstanding, it will be apparent that certain changes andmodifications may be practiced within the scope of the appended claims.For example, lipophilic side chains could be used in practicing themethods of the current invention. Therefore, the above describedembodiments should be considered illustrative and not restrictive andthe instant invention is not limited to the details given herein but maybe modified within the scope of the appended claims.

All publications cited herein are incorporated by reference in theirentireties.

We claim:
 1. An antimicrobial sulfonamide derivative, or a salt or ahydrate thereof, comprising: a core cyclic peptide or core antibiotic ofan acidic lipopeptide antibiotic; and a lipophilic moiety, wherein saidlipophilic moiety is covalently attached to the core cyclic peptide orcore antibiotic via a linking chain which includes a sulfonamide linkageand wherein said core cyclic peptide or core antibiotic is not oflaspartomycin.
 2. The antimicrobial sulfonamide derivative, salt orhydrate of claim 1 in which the linking chain is a sulfonamide linkage.3. The antimicrobial sulfonamide derivative, salt or hydrate of claim 1in which the linking chain is a linker that links the core cyclicpeptide or core antibiotic to the lipophilic moiety.
 4. Theantimicrobial sulfonamide derivative, salt or hydrate of claim 1 whichis a compound according to structural Formula (I):Y—X—N(R⁴)(-L—X—N(R¹))_(m)—R  (I) wherein: Y is a lipophilic moiety; eachX is independently selected from the group consisting of —CO—SO₂—, —CS—,—PO—, —OP(O)—, —OC(O)—, —NHCO and —N(R¹)CO— with the proviso that atleast one X is —SO₂—; M is 0 or 1; L is a linker; N is nitrogen; R¹ andR⁴ are each independently selected from the group consisting ofhydrogen, (C₁-C₂₅) alkyl optionally substituted with one or more of thesame or different R² groups, (C₁-C₂₅) heteroalkyl optionally substitutedwith one or more of the same or different R² groups, (C₅-C₃₀) aryloptionally substituted with one or more of the same or different R²groups, (C₅-C₃₀) arylaryl optionally substituted with one or more of thesame or different R² groups, (C₅-C₃₀) biaryl optionally substituted withone or more of the same or different R² groups, five to thirty memberedheteroaryl optionally substituted with one or more of the same ordifferent R² groups, (C₆-C₃₀) arylalkyl optionally substituted with oneor more of the same or different R² groups and six to thirty memberedheteroarylalkyl optionally substituted with one or more of the same ordifferent R² groups; each R² is independently selected from the groupconsisting of —OR³, —SR³, —NR³R³, —CN, —NO₂, —N₃, —C(O)OR³, —C(O)NR³R³,—C(S)NR³R³, —C(NR³)NR³R³, —CHO, —R³CO, —SO₂R³, —SOR³, —PO(OR³)₂,—PO(OR³), —CO₂H, —SO₃H, —PO₃H, halogen and trihalomethyl; each R³ isindependently selected from the group consisting of hydrogen, (C₁-C₆)alkyl, (C₅-C₁₀) aryl, five to sixteen membered heteroaryl, (C₆-C₁₆)arylalkyl and six to sixteen membered heteroarylalkyl; and R is a corecyclic peptide or core antibiotic of an acidic lipopeptide antibiotic,wherein said core cyclic peptide or core antibiotic is not oflaspartomycin.
 5. The antimicrobial sulfonamide derivative of claim 4 inwhich R is the core cyclic peptide of zaomycin, crystallomycin,aspartocin, amphomycin, glumamycin, brevistin, cerexin A, cerexin B,Antibiotic A-30912, Antibiotic A-1437, Antibiotic A-54145, AntibioticA-21978C or tsushimycin.
 6. The antimicrobial sulfonamide derivative ofclaim 4 in which R is the core antibiotic of zaomycin, crystallomycin,aspartocin, amphomycin, glumamycin, brevistin, cerexin A, cerexin B,Antibiotic A-30912, Antibiotic A-1437, Antibiotic A-54145, AntibioticA-21978C or tsushimycin.
 7. The antimicrobial sulfonamide derivative ofclaim 4 in which R is the core cyclic peptide of aspartocin, AntibioticA*-30912, Antibiotic A-1437, Antibiotic A-54145 or Antibiotic A-21978C.8. The antimicrobial sulfonamide derivative of claim 4 in which R is thecore antibiotic of aspartocin, Antibiotic A-30912, Antibiotic A-1437,Antibiotic A54145 or Antibiotic A-21978C.
 9. The antimicrobialsulfonamide derivative of claim 4 in which R is the core cyclic peptideof aspartocin.
 10. The antimicrobial sulfonamide derivative of claim 4in which R is the core antibiotic of aspartocin.
 11. The antimicrobialsulfonamide derivative of claim 4 in which m is
 1. 12. The antimicrobialsulfonamide derivative of claim 4 in which R¹ and R⁴ are hydrogen. 13.The antimicrobial sulfonamide derivative of claim 4 in which L isselected from the group consisting of:

or a pharmaceutically acceptable salt or hydrate thereof, wherein: n is0, 1, 2 or 3; each S¹ is independently selected from the groupconsisting of hydrogen, (C₁-C₁₀) alkyl optionally substituted with oneor more of the same or different R⁵ groups, (C₁-C₁₀) heteroalkyloptionally substituted with one or more of the same or different R⁵groups, (C₅-C₁₀) aryl optionally substituted with one or more of thesame or different R⁵ groups, (C₅-C₁₅) arylaryl optionally substitutedwith one or more of the same or different R⁵ groups, (C₅-C₁₅) biaryloptionally substituted with one or more of the same or different R⁵groups, five to ten membered heteroaryl optionally substituted with oneor more of the same or different R⁵ groups, (C₆-C₁₆) arylalkyloptionally substituted with one or more of the same or different R⁵groups and six to sixteen membered heteroarylalkyl optionallysubstituted with one or more of the same or different R⁵ groups; each R⁵is independently selected from the group consisting of —OR⁶, —SR⁶,—NR⁶R⁶, —CN, —NO₂, —N₃, —C(O)OR⁶, —C(O)NR⁶R⁶, —C(S)NR⁶R⁶, —C(NR⁶)NR⁶R⁶,—CHO, —R⁶CO, —SO₂R⁶, —SOR⁶, —PO(OR⁶)₂, —PO(OR⁶), —CO₂H, —SO₃H, —PO₃H,halogen and trihalomethyl; each R⁶ is independently selected from thegroup consisting of hydrogen, (C₁-C₆) alkyl, (C₅-C₁₀) aryl, five tosixteen membered heteroaryl, (C₆-C₁₆) arylalkyl and six to sixteenmembered heteroarylalkyl; and each K is independently selected from thegroup consisting of oxygen, nitrogen and sulfur.
 14. The antimicrobialsulfonamide of claim 13 in which each S¹ is independently a side-chainof a genetically encoded α-amino acid.
 15. The antimicrobial sulfonamideof claim 13 in which L is:


16. The antimicrobial sulfonamide derivative of claim 15 in which eachS¹ is independently a side-chain of a genetically encoded α-amino acid.17. The antimicrobial sulfonamide derivative of claim 15 in which n is0.
 18. The antimicrobial sulfonamide derivative of claim 17 in which S¹is hydrogen, Y is decan-yl and R is the core cyclic peptide ofaspartocin.
 19. The antimicrobial sulfonamide derivative of claim 17 inwhich S¹ is —CH₂—CO₂H, —CH₂—CH₂—CO₂H, —C(OH)H—CONH₂, —CH₂—CONH₂ or—CH₂-CH₂—CONH₂ or a salt or hydrate thereof.
 20. The antimicrobialsulfonamide derivative of claim 17 in which S¹ is —CH₂-indol-2-yl or—CH₂-phenyl.
 21. The antimicrobial sulfonamide derivative of claim 13 inwhich L is:

wherein S² and S³ are each independently a side chain of a geneticallyencoded α-amino acid.
 22. The antimicrobial sulfonamide derivative ofclaim 21 in which S² is hydrogen, —CH₂-indol-2-yl, —CH₂—CONH₂ or—CH₂—CH₂—CONH₂ and S³ is —CH₂—CO₂H, —CH₂—CH₂—CO₂H or a salt or hydratethereof.
 23. The antimicrobial sulfonamide derivative of claim 21 inwhich S² is —CH₂—CO₂H, —CH₂—CH₂—CO₂H or a salt or hydrate thereof and S³is —C(OH)H—CONH₂.
 24. The antimicrobial sulfonamide derivative of claim13 in which L is:

wherein S², S³, and S⁴ are each independently a side chain of agenetically encoded α-amino acid.
 25. The antimicrobial sulfonamidederivative of claim 24 in which S² is —CH₂-indol-2-yl, S³ is —CH₂—CONH₂or —CH₂—CH₂—CONH₂ and S⁴ is —CH₂—CO₂H, —CH₂—CH₂—CO₂H or a salt orhydrate thereof.
 26. The antimicrobial sulfonamide derivative of claim24 in which S² is —CH₂-indol-2-yl, S³ is —CH₂—CO₂H, CH₂—CH₂—CO₂H or asalt or hydrate thereof and S⁴ is —CH₂—CONH₂, —CH₂—CH₂—CONH₂ or—C(OH)H—CONH₂.
 27. The antimicrobial sulfonamide derivative of claim 4in which m is
 0. 28. The antimicrobial sulfonamide derivative of claim27 in which R⁴ is hydrogen.
 29. The antimicrobial sulfonamide derivativeof claim 27 which R is the core antibiotic of aspartocin.
 30. Theantimicrobial sulfonamide derivative of claim 27 in which R is the corecyclic peptide of aspartocin.
 31. A pharmaceutical compositioncomprising an antimicrobial sulfonamide derivative according to any oneof claims 1 to 5 and a pharmaceutically acceptable adjuvant, excipient,carrier or diluent.
 32. A method for treating or preventing a microbialinfection, said method comprising the step of administering to a subjecta therapeutically effective amount of a pharmaceutical compositionaccording to claim
 31. 33. A method of inhibiting microbial growth, saidmethod comprising the step of administering to a microbe anantimicrobially effective amount of a pharmaceutical compositionaccording to claim
 31. 34. A method for making an antimicrobialsulfonamide derivative comprising sulfonylating a core antibiotic orcore cyclic peptide with a lipophilic sulfonyl derivative, therebyproviding an antimicrobial sulfonamide derivative.
 35. The method ofclaim 34 in which the lipophilic sulfonyl derivative is an activatedlipophilic sulfonyl ester or a lipophilic sulfonyl halide.
 36. Themethod of claim 35 in which the activated lipophilic sulfonyl ester is alipophilic hydroxybenzotriazole ester.
 37. The method of claim 35 inwhich the lipophilic sulfonyl halide is a lipophilic sulfonyl chloride.38. A method for making an antimicrobial sulfonamide derivativecomprising: sulfonylating a linker with a lipophilic sulfonyl compound,thereby providing a lipophilic sulfonamide linker; and covalentlyattaching the lipophilic sulfonamide linker to a core antibiotic or corecyclic peptide wherein said core cyclic peptide or core antibiotic is ofan acidic lipopeptide antibiotic, thereby yielding an antimicrobialsulfonamide derivative.
 39. A method for making an antimicrobialsulfonamide derivative comprising: covalently attaching a linker to acore antibiotic or core cyclic peptide, thereby providing an linker coreantibiotic or linker core cyclic peptide; and sulfonylating the linkercore antibiotic or linker core cyclic peptide with a lipophilic sulfonylderivative, thereby yielding an antimicrobial sulfonamide derivative.40. A method for treating or preventing a microbial infection, saidmethod comprising the step of administering to a subject atherapeutically effective amount of an antimicrobial sulfonamidederivative according to any one of claims 1 to
 5. 41. The method ofclaim 40 in which the core cyclic peptide is aspartocin.
 42. The methodof claim 40 in which the core antibiotic is aspartocin.
 43. A method ofinhibiting microbial growth, said method comprising the step ofadministering to a microbe an antimicrobially effective amount of anantimicrobial sulfonamide derivative according to any one of claims 1 to5.
 44. The method of claim 43 in which the core cyclic peptide isaspartocin.
 45. The method of claim 43 in which the core antibiotic isaspartocin.